Compositions and Methods for Identifying Agents Which Modulate PTEN Function and PI-3 Kinase Pathways

ABSTRACT

Methods are provided for the identification, biochemical characterization and therapeutic use of agents which impact PTEN, p53, PI-kinase and AKT mediated cellular signaling.

This invention claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Nos. 60/208,437 and 60/274,167 filed May 30,2000 and Mar. 8, 2001 respectively. The entire disclosures of each ofthe above-identified applications is incorporated by reference herein.

Pursuant to 35 U.S.C. §202(c), it is hereby acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the National Institutes of Health,Grant Nos: R01CA75637.

FIELD OF THE INVENTION

This invention relates to the treatment of neoplastic disease and otherpathological conditions characterized by cellular hyperproliferation andloss of regulated growth and motility. More specifically, this inventionprovides methods for the identification and characterization of agentswhich modulate PTEN, PI-3 kinase and AKT activity.

BACKGROUND OF THE INVENTION

Various scientific and scholarly articles are cited throughout thespecification. These articles are incorporated by reference herein todescribe the state of the art to which this invention pertains.

The reversible phosphorylation of proteins and lipids is critical to thecontrol of signal transduction in mammalian cells and is regulated bykinases and phosphatases (Hunter 1995). The product of the tumorsuppressor gene PTEN/MMAC (hereafter termed PTEN) was identified as adual specificity phosphatase and has been shown to dephosphorylateinositol phospholipids in vivo (Li et al Science 1997, Steck et al 1997,Li et al Cancer Res 1997, Myers et al, 1997, Myers et al 1998, Maehamaet al, 1998, Stambolic et al 1998, Wu et al 1998). The PTEN gene, whichis located on the short arm of chromosome 10 (10q23), is mutated in40-50% of high grade gliomas as well as many other tumor types,including those of the prostate, endometrium, breast, and lung (Li etal, Science 1997, Steck et al 1997, Maier et al 1998). In addition, PTENis mutated in several rare autosomal dominant cancer predispositionsyndromes, including Cowden disease, Lhermitte-Duclos disease andBannayan-Zonana syndrome (Liaw et al 1997, Myers et al AJHG 1997,Maehama et al TCB 1999, Cantley and Neel 1999). Furthermore, thephenotype of PTEN-knockout mice revealed a requirement for thisphosphatase in normal development and confirmed its role as a tumorsuppressor (Podsypanina et al PNAS 1999, Suzuki et al Curr Biol 1998, DiChristofano et al Nat Gen 1998).

PTEN is a 55 kDa protein comprising an N-terminal catalytic domain,identified as a segment with homology to the cytoskeletal protein tensinand containing the sequence HC(X)₅R, which is the signature motif ofmembers of the protein tyrosine phosphatase family, and a C-terminal C2domain with lipid-binding and membrane-targeting functions (Lee et alCell 1999). The sequence at the extreme C-terminus of PTEN is similar tosequences known to have binding affinity for PDZ domain-containingproteins. PTEN is a dual specificity phosphatase that displays apronounced preference for acidic substrates (Myers et al PNAS 1997).Importantly, PTEN possesses lipid phosphatase activity, preferentiallydephosphorylating phosphoinositides at the D3 position of the inositolring. It is one of two enzymes known to dephosphorylate the D3 positionin inositol phospholipids.

Since solid tumor progression is dependent on the induction ofangiogenic signals and augmented angiogenesis contributes to the highmortality associated with many cancers, there is a need to elucidate thecellular components that participate in these processes. The urgency ofsuch investigations is underscored by the fatal nature of highlymalignant brain tumors and the fact that the degree of tumorinvasiveness is directly correlated with enhanced angiogenesis.Furthermore, elucidation of cellular components that contribute to theangiogenic switch facilitates the identification of therapeutic agentsand delivery methods useful for the treatment of such malignantdiseases.

PTEN phosphatase activity has also been implicated in many cellularbiochemical reactions. It is an object of the invention to also providemethods for the identification of agents which impact PTEN modulation ofimmunoreceptors, AKT, PI3 kinase and p53 signaling. Methods of use ofagents so identified are also within the scope of the invention.

SUMMARY OF THE INVENTION

PTEN is a pivotal signaling molecule which modulates a wide variety ofcellular processes. These cellular processes include angiogenesis,cellular migration, immunoreceptor modulation, p53 signaling andapoptotic cell death, PI3 and AKT signaling. Mutations in PTEN have beenassociated with the highly malignant progression of brain tumors. Ahallmark of this malignant progression is a dramatic increase inangiogenesis and invasiveness mediated by the concomitant formation ofnew blood vessels.

Thus, in accordance with the present invention methods for the treatmentof cancer associated with PTEN mutation are provided. Exemplary methodsinclude delivery of a native PTEN encoding nucleic acid to cancer cellssuch that the native PTEN protein is expressed. Additional methods forthe treatment of cancer in accordance with the present invention entailthe administration of at least one agent selected from the groupconsisting of PTEN agonists, PI3 kinase inhibitors and AKT inhibitors.The aforementioned treatment protocols may also comprise theadministration of conventional chemotherapeutic agents.

In another aspect of the invention, methods for the prevention ofaberrant angiogenesis are also provided. Aberrant angiogenesis isassociated with several diseases. These include not only cancer, butautoimmune disease, arthritis, systemic lupus erthymatosis, inflammatorybowel disease, coronary artery disease, cerebrovascular disease, andatherosclerosis. Methods for the administration of at least one agentselected from the group consisting of native PTEN encoding nucleicacids, PTEN agonists, PI3 kinase inhibitors and AKT inhibitors for theinhibition or prevention of aberrant angiogenesis are also disclosedherein.

PTEN has also been implicated in immunoreceptor modulation. Thus, in yetanother aspect of the invention, methods for inhibiting the immuneresponse in target cells are provided. PTEN agonists, PI3 kinaseinhibitors and/or AKT inhibitors are administered to patients to preventor inhibit immunoreceptor signaling. Such agents should have efficacy inthe treatment of graft rejection or graft versus host disease.

In yet another aspect of the invention, methods for regulating p53mediated gene expression are also provided. Such methods entail theadministration of native PTEN, PTEN agonists and/or PI3 kinaseinhibitors or AKT inhibitors to induce functional p53 in tumor cells.Such agents effectively increase chemosensitity and/or radiosensitivityof tumor cells by stimulating p53 mediated apoptotic cell death.

Given the widespread effects of PTEN, methods for identifying agentswhich modulate PTEN activity are also provided. Exemplary assays includethose which assess alterations in activated AKT levels, alterations inmicrovessel formation, alterations in TSP1 levels, alterations in VEGFlevels, alterations in TIMP3 levels, alterations in MMP9 activation andalterations PTEN phosphatase activity levels in the presence and absenceof such test agents.

Also provided in accordance with the present invention are highthroughput screening methods for identifying small molecules which haveaffinity for PTEN or fragments thereof. Small molecules so identifiedare within the scope of the present invention and may optionally befurther characterized in the functional assays described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a Western blot and a graph showing that stableexpression of PTEN and PTEN mutants in U87MG cells regulates AKT. (FIG.1A) Cell lysates from the U87MG (U87) cell line and U87 cells infectedwith a retroviral vector encoding PTEN (pBabe-Puro-PTEN) or mutants ofPTEN (pBabe-Puro-PTEN-G129E or R130M) were resolved by SDS-PAGE, equalamounts of proteins were loaded per lane and immunoblotted with antiserato PTEN, phospho-AKT and total AKT, and visualized by enhancedchemiluminescence. The basal levels of PTEN (top), phosphorylated AKT(ser 473) (middle) and total AKT (bottom) are shown. The status of thePTEN gene in each stable cell line was designated as: WT.E1 and WT.C7two separate clones expressing wild type PTEN. R130M and G129E aremutated PTEN proteins. R130M is inert as both a protein and a lipidphosphatase. The G129E PTEN can dephosphorylate acidic phosphopeptides,but cannot dephosphorylate the lipid substrate, PIP3. The U87MG (U87)cell line is the parental cell line isolated from a human glioblastomamultiforme patient. (FIG. 1B) Comparison of in vitro growth of U87MGcells transduced with mutants of PTEN. Equal number of cells (1×105)were incubated in RPMI+10% FBS for different times and cell numbers werequantitated by direct cell counting.

FIGS. 2A, 2B and 2C present data showing the effects of PTEN on growthof U87MG cells in vivo. (FIG. 2A) Cell growth in vivo. In order todetermine the rate of cell growth in vivo, equal amount of cells (5×10⁶)from each cell line were implanted at the right ventral flank bysubcutaneous injection (see legend). The formation and growth of thesubcutaneous tumor was monitored and the volume of the tumor wasdetermined by a three dimensional measurement at the times indicated(day 0, the date of implantation, no tumor is detected). Data wereanalyzed by Student's t-test and differences were significant comparingthe PTEN deficient (U87MG, R130M, G129E) to the wild type PTEN (WT.E1,WT.C7), n=5, number of mice p<0.0001 (FIG. 2B) Stereophotography ofsubcutaneous tumor sites in mice implanted with the parental U87 tumor,PTEN minus (left panel) versus wild type PTEN reconstituted tumor cells(right panel). These tumors represent 25 and 42 days after implantationfor PTEN minus versus wild type PTEN reconstituted tumors, respectively.(FIG. 2C) Immunoblot of cryostat tissue sections from subcutaneous tumorfor the expression pattern of PTEN, AKT and phosphorylated AKT. Frozentissue sections were solubilized in Laemmli sample buffer, total proteinwas quantitated and equal protein was loaded on SDS PAGE. The data shownare representative of tissue analysis from 5 animals per experimentalgroup.

FIGS. 3A-3E show that PTEN suppresses angiogenesis. Immunohistochemicalanalysis of staining with CD31 antibody to evaluate the angiogenesisresponse within the parental U87MG tumor (FIG. 3A) and PTENreconstituted tumors (FIG. 3B) implanted into the subcutaneous tissue.In PTEN minus and tumors expressing mutants of PTEN, there are more newvessels formed (angiogenesis) (upper panel, arrow indicated) than inwild-type PTEN reconstituted tumor (lower panel), indicating the PTENhas direct influence on angiogenesis during tumor growth. (FIG. 3C)Microvessel density (MVD) counts were performed on tumor tissue stainedwith anti-CD31 antibody to determine effect of expression of PTEN andspecific PTEN mutants (G129E or R130M) on tumor induced angiogenesis.Bars represent standard deviation, 5 animals per group. Statisticalanalysis by Student's t-test demonstrate significant difference betweenMVD of PTEN null and PTEN catalytic mutants as compared to wild typePTEN reconstituted tumors, n=5, number of mice p<0.001. (FIG. 3D) PTENregulates the expression of thrombospondin-1 (TSP-1) in U87MG cells.RNAase protection assay was used to measure levels of TSP-1 mRNA in wildtype PTEN expressing U87 cells or cells transduced with a mutantcatalytically dead PTEN (G129R). U87MG cells were infected withretrovirus encoding wild type PTEN (WT), the catalytically dead, G129Rmutant (GR) or empty vector retrovirus (−) and selected for 10 days inpuromycin. RNA was harvested and RNAase protection assays were carriedout using probes for TSP-1 and GAPDH. A probe for glyceraldehydephosphate dehydrogenase (GAPDH) was used as a normalization control.(FIG. 3E) Thrombospondin immunoblot analysis. U87MG transduced with wildtype PTEN (WT) or a catalytic mutant PTEN (G129R) in an ecdysoneinducible expression system were induced (48 hours) with 0.5 μMmuristirone or assayed without induction to determine the effect of PTENexpression on the induction of TSP-1 by Western blotting. Supernatantsfrom cells were prepared and proteins resolved on SDS-PAGE and probedwith anti-TSP-1 antibody. There is clear up-regulation of TSP-1 in wildtype PTEN transduced U87 cultures compared to U87 cells expressing thelipid phosphatase deficient G129R mutant PTEN.

FIG. 4 is a Western blot showing the effect of constitutive PTENreconstitution on VEGF expression in U87MG cells. VEGF immunoblotanalysis of parental U87MG or PTEN wild type or mutant reconstitutedtumor cell lysates revealed a dramatic suppression of VEGF in wild typeand G129E mutant PTEN reconstituted tumor cells.

FIGS. 5A-E are a series of micrographs and a graph showing the effectsof PTEN reconstitution on survival in an orthotopic brain tumor model.Equivalent number of parental U87 cells or U87 cells reconstituted withwild type or mutant alleles of PTEN (see legend) (1×106 cells) wereimplanted in right frontal lobe of nude mice. Stereophotography of wholebrains from mice implanted with U87MG tumor cells (day 25) (FIG. 5A), orPTEN reconstituted (day 42) (FIG. 5B). The implantation site is shown byposition of arrow in the wild type PTEN reconstituted tumor, (FIG. 5C) AR130M PTEN reconstituted tumor implanted into a nude mouse brain(magnification×20). (FIG. 5D) A G129E PTEN reconstituted tumor implantedinto a nude mouse brain. (FIG. 5E) Survival plots for mice implantedwith PTEN minus or parental U87 cells transduced with mutants of PTEN asshown. Survival data represents 15 animals per experimental group. n=15,p<0.0001 for difference observed between the PTEN+ and PTEN− groups forsurvival.

FIG. 6 is a blot showing that PTEN induces the expression of TIMP-3.PTEN regulates the expression of tissue inhibitor of metalloproteinase(TIMP-3) in U87MG cells. RNAase protection assay was used to measurelevels of TIMP-3 mRNA in wild type PTEN expressing U87 cells or cellstransduced with a mutant catalytically dead PTEN (G129R). U87MG cellswere infected with retrovirus encoding wild type PTEN (WT), thecatalytically dead, G129R mutant (GR) or empty vector retrovirus (−) andselected for 10 days in puromycin. RNA was harvested and RNAaseprotection assays were carried out using probes for TIMP-3 and GAPDH. Aprobe for glyceraldehyde phosphate dehydrogenase (GAPDH) was used as anormalization control.

FIG. 7 is a gel showing that PTEN suppresses MMP-9 collagenolyticactivity in vivo. Reverse zymography was used to evaluate the effect ofPTEN reconstitution on collagenolytic activity within tumor tissue. Theenzymatic activities of MMP-2 and MMP-9 were detected based on molecularweight and Western blot analysis (data not shown).

FIG. 8 is a graph showing the effects of PTEN reconstitution on tumorinvasion. Equivalent numbers of parental U87 cells or U87 cellsreconstituted with wild type or mutant forms PTEN. A transwell systemcoated with Matrigel (10 ug/ml) was used to assess the invasiveproperties of U87 cells versus PTEN reconstituted U87 cells in vitro.These data demonstrated that PTEN regulates the capacity of tumor cellsto invade a complex matrix barrier.

FIGS. 9A and 9B are a graph and a blot showing that dominant negativeSyk inhibits phagocytosis The phagocytosis of IgG sensitized sRBCs byJ774A.1 was measured in cells infected with empty vector recombinantvaccinia virus or virus containing dominant negative Syk (D/N Syk). Thecells were infected with the respective viruses for 4 h at 37° C. with5% CO₂, after which they were subjected to IgG sensitized sRBCs in freshmedium at a target to effector ratio equal to 100:1 for 2 h at 37° C.with 5% CO₂. Nonengulfed sRBCs were lysed by water shock and the cellswere fixed and stained with Wright-Giemsa staining before counting thephagocytic index. (FIG. 9A) Quantitation of phagocytosis of IgG coatedsRBCs by J774A.1 cells overexpressing D/N Syk. The columns indicatephagocytic index of uninfected J774A.1 cells, cells infected withvaccinia virus containing vector only and cells infected with vacciniavirus containing D/N Syk. (FIG. 9B) J774A.1 cells infected with vacciniavirus containing D/N Syk expressed D/N Syk protein as shown in lane 3,while lane 1 represents untreated J774A.1 cells and lane 2 representsJ774A.1 cells infected with empty vector vaccinia virus as a control.The error bars represent standard deviation of the mean.

FIGS. 10A and 10B are graphs showing that Src and PI-3 kinase arerequired for ITAM signaling. Cells were treated with PP1, an inhibitorof Src family kinases, at concentrations of 10, 5 and 1 μM orwortmannin, an inhibitor of PI-3 kinase, at concentrations of 10, 5 and1 μg/ml along with an appropriate DMSO control for 1 h in DMEM with 10%FCS and then sensitized sRBCs were added at target to effector ratioequal to 100:1. (FIG. 10A) PP1 blocks the phagocytosis, significantly at10 μM concentration and the effect is dose-dependent. (FIG. 10B)Wortmannin blocks phagocytosis significantly at 5 μg/ml. The columnsindicate phagocytic index of untreated J774A.1 cells treated with DMSO(control), PP1, or wortmannin. The error bars represent standarddeviation of mean.

FIGS. 11A and 11B are blots showing the effect of a dominant negativeSyk and Src inhibitor, PP1, on tyrosine phosphorylation of Cbl inresponse to ITAM stimulation. FIG. 11A, upper panel shows the effects ofD/N Syk on tyrosine phosphorylation of Cbl in response to stimulationwith sensitized sRBCs. Lysates prepared from resting cells or cellsstimulated with sRBCs for 5 minutes were immunoprecipitated withpolyclonal anti-Cbl Ab and immunoblotted with anti-phosphotyrosineantibody. Lane 2 represents Cbl immunoprecipitated from resting J774A.1cells while lanes 5 and 8 represent Cbl immunoprecipitated from restingJ774A.1 cells infected with vaccinia virus containing plain vector ordominant negative Syk respectively. Lane 3 represents Cbl IP from cellsstimulated with sRBCs while lane 6 and 9 represents Cbl IP from cellsinfected with vaccinia virus containing plain vector or dominantnegative Syk respectively stimulated with sRBCs.(FIG. 11B, Upper panel)Cells were treated with PP1 to evaluate the role of the Src familykinases in Cbl tyrosine phosphorylation following stimulation withsensitized sRBCs. Lysates prepared from resting cells or cellsstimulated with sRBCs for 5 minutes were immunoprecipitated withpolyclonal anti-Cbl Ab and the resultant Cbl immunoprecipitates (Cbl IP)were immunoblotted with anti-phosphotyrosine antibody. Lane 2 representsCbl IP from resting J774A.1 cells while lane 5 represents Cbl IP fromresting J774A.1 cells treated with 10 μM PP1. Lane 3 represents Cbl IPfrom cells stimulated with sRBCs while lane 6 represents Cbl IP fromcells treated with PP1 and stimulated with sRBCs. (FIGS. 11A and 11B,lower panels) Anti-Cbl immunoblot of Cbl IP. Lysates prepared fromresting cells or cells stimulated with sRBCs for 5 minutes wereimmunoprecipitated with anti-Cbl antisera and immunoblotted withanti-Cbl antisera. Lanes are as designated in (A).

FIG. 12 is a graph showing that overexpression of PTEN in COST cellsinhibits ITAM signaling. Shows phagocytosis of IgG sensitized sRBCs byCOS cells transfected with FcγRIIa receptor, Syk, Cbl, PTEN or a trapmutant (C124S) of PTEN. The cells were transfected with episomalplasmids containing Syk, Cbl and/or PTEN for 12 h at 37° C. with 5% CO₂,after which were subjected to IgG sensitized sRBCs in fresh medium at atarget to effector ratio equal to 100:1 for two h at 37° C. with 5% CO₂.All experimental groups were transfected with FcRγIIA, Syk, and Cbl,data for Syk and Cbl, not shown. Nonengulfed sRBCs were lysed by watershock and the cells were fixed and stained with Wright-Giemsa stainingbefore counting the phagocytic index. A graphic representation of theinhibition of phagocytosis of IgG coated sRBCs by COST cellsoverexpressing PTEN is shown. The error bars represent standarddeviation of the mean. The red columns indicate the phagocytic index ofplain J774A.1 cells transfected with plasmid containing vector only orPTEN. The green bars represent effect of PTEN on the percent of cellsphagocytic for at least one SRBC.

FIG. 13 is a Western blot showing that PTEN regulates phospho-AKTlevels. A muristirone inducible expression system was used to expressPTEN or PTEN mutants in U87MG cells. Immunoblots of lysates derived fromU87 cells +/− induction for expression of wild type PTEN, wild typeHA-tagged PTEN, or G129R (GR) PTEN mutant of PTEN. were probed withantibodies specific for phospho-(S473)AKT or AKT.

FIG. 14 is a graph and a blot showing the effect of PTEN induction onp53 transcription. U87MG cells expressing wild type PTEN, G129E or G129Rmutants of PTEN under the control of muristirone (Western blot, insert)were transiently cotransfected with pRSVβ-galactosidase and mdm2luciferase. U87MG cells expressed similar levels of PTEN and G129R andslightly higher levels of G129E PTEN under control of a muristirone (+indicates muristirone added to cultures 24 hours prior to transfectionof reporter plasmids; lanes correspond to columns of bar graph).Induction of p53 dependent transcription was quantitated using βgalactosidase as an internal control for transfection efficiency.

FIG. 15 is a graph showing that PI-3 kinase inhibitors block tumorgrowth. Tumor volume was measured in DMSO treated mice or LY294002 (aPI3 kinase inhibitor) treated (100 mg/kg/day×2 weeks) mice; treatmentwas concomitant with tumor implantation.

FIG. 16 is a graph showing quantitation of CD31 positive microvesselswithin tumor tissue in the presence and absence of LY294002. Note thedramatic inhibitory effect of LY294002 on tumor-induced angiogenesis.Bars represent standard deviation of the mean (p<0.001).

FIG. 17 is a graph showing that administration of LY294002 dramaticallyreduces the incidence of brain tumors.

FIG. 18 is a graph showing that the effect of LY294002 onchemosensitivity of glioma cells. −/−, no addition; −/1 mcM VP16, U87MGglioma cells exposed to 1 mcM VP16 only; −/5 mcM VP16, cells exposed to5 mcM VP16 only; LY/1 mcMVP16, cells exposed to 10 uM LY294002+1 mcMVP16; LY/5 mcM VP16, cells exposed to 10 uM LY294002+5 mcM VP16. Cellswere incubated for 48 hours with above components prior to MTT analysisfor viable cell numbers. Bars are standard deviation of meanobservation.

FIG. 19 is a graph showing the kinetic effect of LY294002 on EtoposideChemosensitivity. −/−, no addition; LY294002/−, U87MG cells exposed to10 uM LY294002 alone; −/0.5 mcM VP16, cells exposed to 0.5 mcM VP16alone; −/1 mcM VP16, cells exposed to 1 mcM VP16 alone; −/5 mcM VP16,cells exposed to 5 mcM VP16 alone; LY/0.5 mcM VP16, cells exposed to 10uM LY294002+0.5 mcM VP16; LY/1 mcM VP16, cells exposed to 10 uMLY294002+1 mcM VP16; LY/5 mcM VP16, cells exposed to 10 uM LY294002+5mcM VP16. Cells were assayed by MTT at different times after addition ofLY294002 and/or VP16 as shown.

FIGS. 20A and 20B depict a PTEN encoding nucleic acid (SEQ ID NO: 1) andthe amino acid sequence (SEQ ID NO: 2) of PTEN, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Tumor progression, particularly in aggressive and malignant tumors, isassociated with the induction of angiogenesis, a process termed theangiogenic switch. Mutations of the tumor suppressor PTEN, a phosphatasewith specificity for D3 phosphorylated inositol phospholipids, areassociated with malignant and invasive tumor progression. PTEN is,therefore, a critical regulator of tumor progression that acts bymodulating the angiogenic switch response.

To address the role of PTEN in the angiogenic switch response, acritical predictor of the metastatic potential of a tumor, a modelsystem was developed utilizing the U87MG glioma cell line. The U87MGcell line, which is null for PTEN, is a highly metastatic cell linederived from a human glioblastoma multiforme patient. U87MG glioma cellsstably reconstituted with PTEN cDNA were tested for growth in a nudemouse orthotopic brain tumor model. The introduction of wild type PTENinto U87MG cells results in decreased tumor growth in vivo and prolongedsurvival of mice implanted intracranially with these cells. PTENreconstitution diminished phosphorylation of AKT within thePTEN-reconstituted tumor, induced thrombospondin 1 expression, andsuppressed VEGF expression and angiogenic activity. These effects werenot observed in tumors reconstituted with the G129E mutant form of PTEN,in which lipid phosphatase activity is ablated. These data provide thefirst direct evidence that PTEN coordinately regulates the angiogenicswitch and the progression of gliomas to a malignant phenotype via theregulation of phosphoinositide-dependent signals which control p53transcriptional activity.

Thus in accordance with the present invention, methods are provided foridentifying and characterizing small molecules which impact PTENmodulated angiogenesis and tumor progression. A variety of biochemicalassays are provided which will facilitate the characterization of suchmolecules. Exemplary assays include those suitable for assessing matrixdegradation, angiogenesis, tumor invasion and suppression of matrixmetalloproteinase 9 activity.

The regulatory role for PTEN phosphatase is widespread throughout thecell. As described in Example III, PTEN also regulates inflammatorysignaling. Immunoreceptor activation is associated with antibodydependent cell mediated toxicity, NK and CTL lysis of target cells, suchas tumor cells, parasitic cells and microorganisms. The data presentedherein indicate that PTEN controls immunoreceptor desensitization invivo. This observation provides the basis for the development of assaysand methods to identify and characterize small molecules which haveefficacy in the treatment immune disorders associated with hyperactiveinflammatory responses. Such molecules should also have efficacy in thetreatment of graft versus host disease and graft rejection. Methods ofuse of agents so identified are also in the scope of the invention. PTENinhibitors should effectively block immunoreceptor desensitizationthereby augmenting the immunotherapeutic activity of immune cells.Immune cells that may be targeted with these inhibitors include T cells,B cells, macrophages, dendritic cells, neutrophils, mast cells,eosinophils, and platelets.

A detailed analysis of the PTEN and PI-3 kinase signaling cascade andits impact on p53 mediated transcription is provided in Example IV. Inaccordance with the present invention, it has been discovered that p53mediated transcription is dependent upon proper PTEN/PI3 kinasesignaling. These data also indicate that PI-3 kinase inhibitors have invivo antiangiogenic activity. It has also been discovered that PTENexerts control over p53 levels in cells as cells that contain mutatedPTEN have a marked reduction in functional p53 levels. Reduced p53function is associated with reduced sensitivity to stress orchemotherapy induced apoptosis. Thus, this data provides the basis forthe development of additional biological assays for assessing theeffects of small molecules which inhibit PTEN/PI3 kinase/p53 signaling.Such small molecules should also have efficacy in the treatment ofcancer.

PTEN activity has also been implicated in chemo- and radio-sensitivityas set forth in Example V. Thus, based on the data presented herein, ithas been discovered that activation of PTEN and the PI3 kinase pathwaysensitizes cells to p53 mediated cell death through the control of p53induced apoptosis. These observations thus provide the basis foradditional methods for identifying efficacious chemotherapeuticcombination therapies which should be effective in the treatment ofcancer.

The following description sets forth the general procedures involved inpracticing the present invention. To the extent that specific materialsare mentioned, it is merely for purposes of illustration and is notintended to limit the invention. Unless otherwise specified, generalcloning and gene expression procedures, such as those set forth inCurrent Protocols in Molecular Biology, Ausubel et al. eds., JW Wileyand Sons, NY (1998)are utilized.

I. Definitions

Various terms relating to the biological molecules of the presentinvention are used hereinabove and also throughout the specificationsand claims.

“Nucleic acid” or a “nucleic acid molecule” as used herein refers to anyDNA or RNA molecule, either single or double stranded and, if singlestranded, the molecule of its complementary sequence in either linear orcircular form. In discussing nucleic acid molecules, a sequence orstructure of a particular nucleic acid molecule may be described hereinaccording to the normal convention of providing the sequence in the 5′to 3′ direction. With reference to nucleic acids of the invention, theterm “isolated nucleic acid” is sometimes used. This term, when appliedto DNA, refers to a DNA molecule that is separated from sequences withwhich it is immediately contiguous in the naturally occurring genome ofthe organism in which it originated. For example, an “isolated nucleicacid” may comprise a DNA molecule inserted into a vector, such as aplasmid or virus vector, or integrated into the genomic DNA of aprokaryotic or eukaryotic cell or host organism.

When applied to RNA, the term “isolated nucleic acid” refers primarilyto an RNA molecule encoded by an isolated DNA molecule as defined above.Alternatively, the term may refer to an RNA molecule that has beensufficiently separated from other nucleic acids with which it would beassociated in its natural state (i.e., in cells or tissues). An isolatednucleic acid (either DNA or RNA) may further represent a moleculeproduced directly by biological or synthetic means and separated fromother components present during its production.

“Natural allelic variants”, “mutants” and “derivatives” of particularsequences of nucleic acids refer to nucleic acid sequences that areclosely related to a particular sequence but which may possess, eithernaturally or by design, changes in sequence or structure. By closelyrelated, it is meant that at least about 75%, but often, more than 90%,of the nucleotides of the sequence match over the defined length of thenucleic acid sequence referred to using a specific SEQ ID NO. Changes ordifferences in nucleotide sequence between closely related nucleic acidsequences may represent nucleotide changes in the sequence that ariseduring the course of normal replication or duplication in nature of theparticular nucleic acid sequence. Other changes may be specificallydesigned and introduced into the sequence for specific purposes, such asto change an amino acid codon or sequence in a regulatory region of thenucleic acid. Such specific changes may be made in vitro using a varietyof mutagenesis techniques or produced in a host organism placed underparticular selection conditions that induce or select for the changes.Such sequence variants generated specifically may be referred to as“mutants” or “derivatives” of the original sequence.

The present invention also includes methods of use for active portions,fragments, derivatives and functional or non-functional mimetics of PTENpolypeptides or proteins of the invention. An “active portion” of PTENpolypeptide means a peptide that is less than the full length PTENpolypeptide, but which retains measurable biological activity.

A “fragment” or “portion” of the PTEN polypeptide means a stretch ofamino acid residues of at least about five to seven contiguous aminoacids, often at least about seven to nine contiguous amino acids,typically at least about nine to thirteen contiguous amino acids and,most preferably, at least about twenty to thirty or more contiguousamino acids. Fragments of the PTEN polypeptide sequence, antigenicdeterminants, or epitopes are useful for eliciting immune responses to aportion of the PTEN amino acid sequence.

A “derivative” of the PTEN polypeptide or a fragment thereof means apolypeptide modified by varying the amino acid sequence of the protein,e.g. by manipulation of the nucleic acid encoding the protein or byaltering the protein itself. Such derivatives of the natural amino acidsequence may involve insertion, addition, deletion or substitution ofone or more amino acids, and may or may not alter the essential activityof the original PTEN polypeptide.

As mentioned above, the PTEN polypeptide or protein of the inventionincludes any analogue, fragment, derivative or mutant which is derivedfrom a PTEN polypeptide and which retains at least one property or othercharacteristic of the PTEN polypeptide. Different “variants” of the PTENpolypeptide exist in nature. These variants may be alleles characterizedby differences in the nucleotide sequences of the gene coding for theprotein, or may involve different RNA processing or post-translationalmodifications. The skilled person can produce variants having single ormultiple amino acid substitutions, deletions, additions or replacements.These variants may include inter alia: (a) variants in which one or moreamino acids residues are substituted with conservative ornon-conservative amino acids, (b) variants in which one or more aminoacids are added to the PTEN polypeptide, (c) variants in which one ormore amino acids include a substituent group, and (d) variants in whichthe PTEN polypeptide is fused with another peptide or polypeptide suchas a fusion partner, a protein tag or other chemical moiety, that mayconfer useful properties to the PTEN polypeptide, such as, for example,an epitope for an antibody, a polyhistidine sequence, a biotin moietyand the like. Other PTEN polypeptides of the invention include variantsin which amino acid residues from one species are substituted for thecorresponding residues in another species, either at the conserved ornon-conserved positions. In another embodiment, amino acid residues atnon-conserved positions are substituted with conservative ornon-conservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques are known to the person having ordinary skillin the art.

To the extent such allelic variations, analogues, fragments,derivatives, mutants, and modifications, including alternative nucleicacid processing forms and alternative post-translational modificationforms result in derivatives of the PTEN polypeptide that retain any ofthe biological properties of the PTEN polypeptide, they are includedwithin the scope of this invention.

The term “functional” as used herein implies that the nucleic or aminoacid sequence is functional for the recited assay or purpose.

The phrase “consisting essentially of” when referring to a particularnucleotide or amino acid means a sequence having the properties of agiven SEQ ID No:. For example, when used in reference to an amino acidsequence, the phrase includes the sequence per se and molecularmodifications that would not affect the basic and novel characteristicsof the sequence.

A “replicon” is any genetic element, for example, a plasmid, cosmid,bacmid, phage or virus, that is capable of replication largely under itsown control. A replicon may be either RNA or DNA and may be single ordouble stranded.

A “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage orvirus, to which another genetic sequence or element (either DNA or RNA)may be attached so as to bring about the replication of the attachedsequence or element.

An “expression operon” refers to a nucleic acid segment that may possesstranscriptional and translational control sequences, such as promoters,enhancers, translational start signals (e.g., ATG or AUG codons),polyadenylation signals, terminators, and the like, and which facilitatethe expression of a polypeptide coding sequence in a host cell ororganism.

The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains 15-25 or morenucleotides, although it may contain fewer nucleotides. The probesherein are selected to be “substantially” complementary to differentstrands of a particular target nucleic acid sequence. This means thatthe probes must be sufficiently complementary so as to be able to“specifically hybridize” or anneal with their respective target strandsunder a set of pre-determined conditions. Therefore, the probe sequenceneed not reflect the exact complementary sequence of the target. Forexample, a non-complementary nucleotide fragment may be attached to the5′ or 3′ end of the probe, with the remainder of the probe sequencebeing complementary to the target strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprobe, provided that the probe sequence has sufficient complementaritywith the sequence of the target nucleic acid to anneal therewithspecfically.

The term “specifically hybridize” refers to the association between twosingle-stranded nucleic acid molecules of sufficiently complementarysequence to permit such hybridization under pre-determined conditionsgenerally used in the art (sometimes termed “substantiallycomplementary”). In particular, the term refers to hybridization of anoligonucleotide with a substantially complementary sequence containedwithin a single-stranded DNA or RNA molecule of the invention, to thesubstantial exclusion of hybridization of the oligonucleotide withsingle-stranded nucleic acids of non-complementary sequence.

Amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form may besubstituted for any L-amino acid residue, provided the desiredproperties of the polypeptide are retained.

All amino-acid residue sequences represented herein conform to theconventional left-to-right amino-terminus to carboxy-terminusorientation.

The term “isolated protein” or “isolated and purified protein” issometimes used herein. This term refers primarily to a protein producedby expression of an isolated nucleic acid molecule of the invention.Alternatively, this term may refer to a protein that has beensufficiently separated from other proteins with which it would naturallybe associated, so as to exist in “substantially pure” form. “Isolated”is not meant to exclude artificial or synthetic mixtures with othercompounds or materials, or the presence of impurities that do notinterfere with the fundamental activity, and that may be present, forexample, due to incomplete purification, addition of stabilizers, orcompounding into, for example, immunogenic preparations orpharmaceutically acceptable preparations.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight of a given material (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-95% by weightof the given compound. Purity is measured by methods appropriate for thegiven compound (e.g. chromatographic methods, agarose or polyacrylamidegel electrophoresis, HPLC analysis, and the like).

The term “tag,” “tag sequence” or “protein tag” refers to a chemicalmoiety, either a nucleotide, oligonucleotide, polynucleotide or an aminoacid, peptide or protein or other chemical, that when added to anothersequence, provides additional utility or confers useful properties,particularly in the detection or isolation, to that sequence. Thus, forexample, a homopolymer nucleic acid sequence or a nucleic acid sequencecomplementary to a capture oligonucleotide may be added to a primer orprobe sequence to facilitate the subsequent isolation of an extensionproduct or hybridized product. In the case of protein tags, histidineresidues (e.g., 4 to 8 consecutive histidine residues) may be added toeither the amino- or carboxy-terminus of a protein to facilitate proteinisolation by chelating metal chromatography. Alternatively, amino acidsequences, peptides, proteins or fusion partners representing epitopesor binding determinants reactive with specific antibody molecules orother molecules (e.g., flag epitope, c-myc epitope, transmembraneepitope of the influenza A virus hemaglutinin protein, protein A,cellulose binding domain, calmodulin binding protein, maltose bindingprotein, chitin binding domain, glutathione S-transferase, and the like)may be added to proteins to facilitate protein isolation by proceduressuch as affinity or immunoaffinity chromatography. Chemical tag moietiesinclude such molecules as biotin, which may be added to either nucleicacids or proteins and facilitate isolation or detection by interactionwith avidin reagents, and the like. Numerous other tag moieties areknown to, and can be envisioned by, the trained artisan, and arecontemplated to be within the scope of this definition.

As used herein, the terms “reporter,” “reporter system”, “reportergene,” or “reporter gene product” shall mean an operative genetic systemin which a nucleic acid comprises a gene that encodes a product thatwhen expressed produces a reporter signal that is a readily measurable,e.g., by biological assay, immunoassay, radioimmunoassay, or bycolorimetric, fluorogenic, chemiluminescent or other methods. Thenucleic acid may be either RNA or DNA, linear or circular, single ordouble stranded, antisense or sense polarity, and is operatively linkedto the necessary control elements for the expression of the reportergene product. The required control elements will vary according to thenature of the reporter system and whether the reporter gene is in theform of DNA or RNA, but may include, but not be limited to, suchelements as promoters, enhancers, translational control sequences, polyA addition signals, transcriptional termination signals and the like.

The terms “transform”, “transfect”, “transduce”, shall refer to anymethod or means by which a nucleic acid is introduced into a cell orhost organism and may be used interchangeably to convey the samemeaning. Such methods include, but are not limited to, transfection,electroporation, microinjection, PEG-fusion and the like. The introducednucleic acid may or may not be integrated (covalently linked) intonucleic acid of the recipient cell or organism. In bacterial, yeast,plant and mammalian cells, for example, the introduced nucleic acid maybe maintained as an episomal element or independent replicon such as aplasmid. Alternatively, the introduced nucleic acid may becomeintegrated into the nucleic acid of the recipient cell or organism andbe stably maintained in that cell or organism and further passed on orinherited to progeny cells or organisms of the recipient cell ororganism. In other manners, the introduced nucleic acid may exist in therecipient cell or host organism only transiently.

A “clone” or “clonal cell population” is a population of cells derivedfrom a single cell or common ancestor by mitosis.

A “cell line” is a clone of a primary cell or cell population that iscapable of stable growth in vitro for many generations.

An “antibody” or “antibody molecule” is any immunoglobulin, includingantibodies and fragments thereof, that binds to a specific antigen. Theterm includes polyclonal, monoclonal, chimeric, and bispecificantibodies. As used herein, antibody or antibody molecule contemplatesboth an intact immunoglobulin molecule and an immunologically activeportion of an immunoglobulin molecule such as those portions known inthe art as Fab, Fab′, F(ab′)2 and F(v).

II. Preparation of PTEN-Encoding Nucleic Acid Molecules, PTEN Proteinsand Antibodies Thereto

The PTEN protein comprises, from amino- to carboxy-terminus, a proteintyrosine phosphatase catalytic domain that has considerable homology tothe cytoskeletal protein tensin, a C2 domain that confers lipid-bindingand membrane-targeting, and a PDZ domain-binding site that contributesto membrane localization and protein stability (Lee et al Cell 1999, Wuet al PNAS 2000;). The amino-terminal catalytic domain includes theHC(X)₅R sequence, which is the signature motif of protein tyrosinephosphatases. The Genbank accession number for the human PTEN encodingnucleic acid molecule is NM000313. The amino acid sequence of PTEN isprovided in FIG. 20B. These sequences are also referred to herein as SEQID NO: 1 and SEQ ID NO: 2.

The PTEN protein, hereafter termed PTEN, is classified as a dualspecificity phosphatase, whose substrate targets include phosphorylatedproteins and inositol phospholipids. PTEN is distinguished by the factthat, unlike other dual specificity phosphatases, it preferentiallydephosphorylates phosphoinositides at the D3 position of the inositolring (Maehama et al Trends Cell Biol. 1999, Maehama et al J Biol Chem1998). PTEN is the product of the tumor suppressor gene PTEN/MMAC,mutations in which have been correlated with a number of different tumortypes, including those of the brain, prostate, endometrium, breast, andlung.

A. Nucleic Acid Molecules

Nucleic acid molecules encoding PTEN may be prepared by two generalmethods: (1) They may be synthesized from appropriate nucleotidetriphosphates, or (2) they may be isolated from biological sources. Bothmethods utilize protocols well known in the art.

The availability of nucleotide sequence information, such as the fulllength cDNA having Sequence I.D. No. 1, enables preparation of anisolated nucleic acid molecule of the invention by oligonucleotidesynthesis. Synthetic oligonucleotides may be prepared by thephosphoramadite method employed in the Applied Biosystems 38A DNASynthesizer or similar devices. The resultant construct may be purifiedaccording to methods known in the art, such as high performance liquidchromatography (HPLC). Long, double-stranded polynucleotides, such as aDNA molecule of the present invention, must be synthesized in stages,due to the size limitations inherent in current oligonucleotidesynthetic methods. Thus, for example, a large double-stranded DNAmolecule may be synthesized as several smaller segments of appropriatecomplementarity. Complementary segments thus produced may be annealedsuch that each segment possesses appropriate cohesive termini forattachment of an adjacent segment. Adjacent segments may be ligated byannealing cohesive termini in the presence of DNA ligase to constructthe entire protein encoding sequence. A synthetic DNA molecule soconstructed may then be cloned and amplified in an appropriate vector.

Nucleic acid sequences encoding PTEN may be isolated from appropriatebiological sources using methods known in the art. In a preferredembodiment, a cDNA clone is isolated from an expression library of humanorigin. In an alternative embodiment, genomic clones encoding PTEN maybe isolated.

Nucleic acids of the present invention may be maintained as DNA in anyconvenient cloning vector. In a preferred embodiment, clones aremaintained in plasmid cloning/expression vector, such as pBluescript(Stratagene, La Jolla, Calif.), which is propagated in a suitable E.coli host cell.

PTEN-encoding nucleic acid molecules of the invention include cDNA,genomic DNA, RNA, and fragments thereof which may be single- ordouble-stranded. Thus, this invention provides oligonucleotides (senseor antisense strands of DNA or RNA) having sequences capable ofhybridizing with at least one sequence of a nucleic acid molecule of thepresent invention, such as selected segments of the cDNA having SequenceI.D. No. 1. Such oligonucleotides are useful as agents to inhibit oraugment PTEN activity in cells or tissues. In particular, the presentinvention describes the use of PTEN encoding nucleic acids forreconstitution of PTEN activity in malignant cells or tissues for thepurposes anti-cancer therapy.

B. Proteins

Full-length PTEN of the present invention may be prepared in a varietyof ways, according to known methods. The protein may be purified fromappropriate sources, e.g., human or animal cultured cells or tissues, byimmunoaffinity purification. However, this is not a preferred method dueto the small amounts of protein likely to be present in a given celltype at any time. The availability of nucleic acids molecules encodingPTEN enables production of the protein using in vitro expression methodsknown in the art. For example, a cDNA or gene may be cloned into anappropriate in vitro transcription vector, such a pSP64 or pSP65 for invitro transcription, followed by cell-free translation in a suitablecell-free translation system, such as wheat germ or rabbitreticulocytes. In vitro transcription and translation systems arecommercially available, e.g., from Promega Biotech, Madison, Wisconsinor BRL, Rockville, Md.

Alternatively, according to a preferred embodiment, larger quantities ofPTEN may be produced by expression in a suitable procaryotic oreucaryotic system. For example, part or all of a DNA molecule, such asthe cDNA having Sequence I.D. No. 1, may be inserted into a plasmidvector adapted for expression in a bacterial cell, such as E. coli, orinto a baculovirus vector for expression in an insect cell. Such vectorscomprise the regulatory elements necessary for expression of the DNA inthe bacterial host cell, positioned in such a manner as to permitexpression of the DNA in the host cell. Such regulatory elementsrequired for expression include promoter sequences, transcriptioninitiation sequences and, optionally, enhancer sequences.

The PTEN produced by gene expression in a recombinant procaryotic oreucaryotic system may be purified according to methods known in the art.In a preferred embodiment, a commercially available expression/secretionsystem can be used, whereby the recombinant protein is expressed andthereafter secreted from the host cell, to be easily purified from thesurrounding medium. If expression/secretion vectors are not used, analternative approach involves purifying the recombinant protein byaffinity separation, such as by immunological interaction withantibodies that bind specifically to the recombinant protein. Suchmethods are commonly used by skilled practitioners.

The PTEN proteins of the invention, prepared by the aforementionedmethods, may be analyzed according to standard procedures. For example,such proteins may be subjected to amino acid sequence analysis,according to known methods.

The present invention also provides methods of use of antibodies capableof immunospecifically binding to proteins of the invention. Polyclonalantibodies directed toward PTEN or fragments thereof may be preparedaccording to standard methods. In a preferred embodiment, monoclonalantibodies are prepared, which react immunospecifically with variousepitopes of PTEN. Monoclonal antibodies may be prepared according togeneral methods of Köhler and Milstein, following standard protocols.Polyclonal or monoclonal antibodies that immunospecifically interactwith PTEN can be utilized for identifying and purifying such proteins.For example, antibodies may be utilized for affinity separation ofproteins with which they immunospecifically interact. Antibodies mayalso be used to immunoprecipitate proteins from a sample containing amixture of proteins and other biological molecules. Other uses ofanti-PTEN antibodies are described below.

III. Uses of PTEN-Encoding Nucleic Acids, PTEN Proteins and AntibodiesThereto

Tumor suppressor proteins constitute a functional family of proteinsknown to be essential regulators of cellular proliferation and, as such,provide suitable targets for the development of therapeutic agents formodulating their activity in a cell. Since PTEN is a tumor suppressorprotein implicated in the etiology of many malignant diseases, methodsfor identifying agents that modulate its activity are provided. Agentsso identified should have efficacy in the treatment of a variety ofmalignant diseases. The use of PTEN modulating agents in conjunctionwith other known anti-cancer treatments such as chemotherapy andradiation therapy is also described. Moreover, such therapeutic agentswill also be useful for modulating the activity of PTEN in othercellular systems. Since there are several diseases, other than cancer,in which abnormal angiogenesis contributes to the etiology of thedisease, the administration of PTEN modulating agents should providetherapeutic advantages for the treatment of these conditions.Utilization of therapeutic agents that modulate PTEN activity could alsobe used effectively to treat disorders characterized hyperactivity ofthe inflammatory immune response.

A. PTEN-Encoding Nucleic Acids

PTEN-encoding nucleic acids may be used for a variety of purposes inaccordance with the present invention. PTEN-encoding DNA, RNA, orfragments thereof may be used as probes to detect the presence of and/orexpression of genes encoding PTEN. Methods in which PTEN-encodingnucleic acids may be utilized as probes for such assays include, but arenot limited to: (1) in situ hybridization; (2) Southern hybridization(3) northern hybridization; and (4) assorted amplification reactionssuch as polymerase chain reactions (PCR).

Nucleic acid molecules, or fragments thereof, encoding PTEN may also beutilized to control the expression of PTEN, thereby regulating theamount of protein available to participate in tumor suppressor signalingpathways. Alterations in the physiological amount of PTEN may actsynergistically with chemotherapeutic agents used to treat cancer. Inone embodiment, the nucleic acid molecules of the invention will be usedto restore PTEN expression to normal cellular levels or overexpress PTENin a population of malignant cells. In this embodiment, reconstitutionof signaling events downstream of PTEN abrogates the aberrant cellularproliferation observed in malignant cells.

In another embodiment, the nucleic acid molecules of the invention maybe used to decrease expression of PTEN in a population of target cells.In this embodiment, oligonucleotides are targeted to specific regions ofPTEN-encoding genes that are critical for gene expression. The use ofantisense oligonucleotides to decrease expression levels of apre-determined gene is known in the art. In a preferred embodiment, suchantisense oligonucleotides are modified in various ways to increasetheir stability and membrane permeability, so as to maximize theireffective delivery to target cells in vitro and in vivo. Suchmodifications include the preparation of phosphorothioate ormethylphosphonate derivatives, among many others, according toprocedures known in the art. The use of antisense oligonucleotides forthe modulation of PTEN expression is disclosed in U.S. Pat. No.6,020,199, filed Feb. 1, 2000, the entire disclosure of which isincorporated by reference.

As described above, PTEN-encoding nucleic acids are also used toadvantage to produce large quantities of substantially pure PTENprotein, or selected portions thereof. In a preferred embodiment, theN-terminal “catalytic domain” of PTEN is produced by expression of anucleic acid encoding the domain. The full-length protein or selecteddomain is thereafter used for various research, diagnostic andtherapeutic purposes, as described below.

B. PTEN Protein and Antibodies

Purified PTEN, or fragments thereof, may be used to produce polyclonalor monoclonal antibodies which also may serve as sensitive detectionreagents for the presence and accumulation of PTEN (or complexescontaining PTEN) in cultured cells or tissues from living patients (theterm “patients” refers to both humans and animals). Recombinanttechniques enable expression of fusion proteins containing part or allof the PTEN protein. The full length protein or fragments of the proteinmay be used to advantage to generate an array of monoclonal antibodiesspecific for various epitopes of the protein, thereby providing evengreater sensitivity for detection of the protein in cells or tissue.

Polyclonal or monoclonal antibodies immunologically specific for PTENmay be used in a variety of assays designed to detect and quantitate theprotein, which may be useful for diagnosing a PTEN-related malignantdisease in a patient. Such assays include, but are not limited to: (1)flow cytometric analysis; (2) immunochemical localization in PTEN incultured cells or tissues; and (3) immunoblot analysis (e.g., dot blot,Western blot) of extracts from various cells and tissues. Additionally,as described above, anti-PTEN can be used for purification of PTEN(e.g., affinity column purification, immunoprecipitation).

Anti-PTEN antibodies may also be utilized as therapeutic agents to blockthe normal functionality of PTEN in a target cell population, such as aninflammatory cell. Thus, similar to the antisense oligonucleotidesdescribed above, anti-PTEN antibodies may be delivered to a target cellpopulation by methods known in the art (i.e. through various lipophiliccarriers that enable delivery of the compound of interest to the targetcell cytoplasm) where the antibodies may interact with intrinsic PTEN torender it nonfunctional.

From the foregoing discussion, it can be seen that PTEN-encoding nucleicacids and PTEN proteins of the invention can be used to modulate PTENgene expression and protein activity for the purposes of assessing theimpact of PTEN modulation on the regulation of proliferative pathways ofa cell or tissue sample. It is expected that these tools will beparticularly useful for the treatment of human neoplastic disease inthat PTEN-encoding nucleic acids, proteins and antibodies are excellentcandidates for use as therapeutic agents.

Although the compositions of the invention have been described withrespect to human therapeutics, it will be apparent to one skilled in theart that these tools will also be useful in animal and cultured cellexperimentation with respect to various malignancies and/or otherconditions manifested by alterations in cellular proliferation. Astherapeutics, they can be used either alone or as adjuncts to otherchemotherapeutic drugs to improve the effectiveness of such anti-canceragents.

III. Therapeutics A. Rational Drug Design

Since PTEN is a tumor suppressor protein implicated in the etiology ofmany malignant diseases, including, but not limited to, those of thebrain, prostate, endometrium, and lung, methods for identifying agentsthat modulate its activity should result in the generation ofefficacious therapeutic agents for the treatment of a variety ofmalignant and inflammatory diseases.

The crystal structure of PTEN, solved in 1999, revealed that the 403amino acid protein comprises three domains of known function. These arethe N terminal catalytic domain(residues 1-185), the C2 domain (residues186-349) that participates in membrane binding and catalysis and the Cterminal tail region (residues 350-403). See FIG. 20. Each of thesedomains provide suitable targets for the rational design of therapeuticagents which modulate PTEN activity. Particularly preferred regions arethe N terminal and C2 domains, specifically regions including certainunique residues within and adjacent to the P loop, the WPD loop and theTI loop. It is these residues that participate in specific PIP3substrate recognition and catalysis thereof. Another suitable regionincludes the C terminal tail which participates in PTEN regulatory anddegradation in vivo. Small peptide molecules corresponding to theseregions may be used to advantage in the design of therapeutic agentswhich effectively modulate the activity of PTEN, P1-3 kinase cascades,AKT cascades, as well as p53-mediated transcription and cell death.

PTEN is phosphorylated on tyrosine, serine and threonine residues.Agents which affect the phosphorylation state of the protein will alsobe screened as those small molecules which affect phosphorylation ofPTEN should also modulate PTEN interactions with other proteins. TheDLDLTYIYP motif (residues 22-30; SEQ ID NO: 3) at the extreme N terminusof PTEN contains a YxxP motif (SEQ ID NO: 4), a possible docking sitefor adapter proteins like crk and crkl via SH2 interactions. Anothermotif, YFSPN (SEQ ID NO: 5) in the C terminus has been identified as thebinding site for crk and crkl. The YLVLTL motif (SEQ ID NO: 6) in theextreme C terminus is a site for SH2 interactions with Shc or SHP-1. TheYSYL motif (SEQ ID NO: 7), which contains a tyrosine at position 178, is100% conserved from Drosophila to man. Other tyrosine phosphorylatedmotifs include: YRNNIDD (SEQ ID NO: 8), Y at position 46, a sequencepresent in the catalytic domain identified as a binding site for Grb2via its SH2 domain.

Binding and inhibition of PTEN phosphatase may be assessed usingrecombinant wild type PTEN or mutants of PTEN and appropriate PIP₃substrates to measure dephosphorylation of PIP₃ at D3 position. Agentswhich modulate PTEN phosphatase action should have efficacy in thetreatment of cancer and inflammatory diseases. The dephosphorylation ofphosphatidylinositol 3,4,5,-trisphosphate (PIP₃) is carried out in areaction mixture consisting of 100 mM Tris-HCl (pH 8), 10 mMdithiothreitol, 0.5 mM diC₁₆ phosphatidylserine (PS), 25 uM PIP₃, diC₁₆,BIOMOL PH-107 (BIOMOL, Inc.) and 50 μg/ml purified recombinant PTEN.Lipids were prepared in organic solvents and dispensed into 1.5 mlmicrofuge tubes followed by solvent removal under reduced pressure.Buffer is then added and a lipid suspension is formed by sonication.PTEN phosphatase assays are initiated by the addition of PTEN andcarried out at 37° C. At different time points, 15 μl of 100 μM NEM(N-ethylmaleimide) is added to 10 μl of reaction mixture followed byrapid centrifugation 18,000×g for 15 minutes at 4° C. Liberatedinorganic phosphate is detected in twenty microliters of supernatantusing the Malachite green assay and an inorganic phosphate standardcurve. Malachite green reaction with inorganic phosphate is detectedspectrophotometrically at 620 nm wavelength. The N terminal domain alsocontains the P-loop (HCKAGKGR, residues (123-130; SEQ ID NO: 9) which isunique to PTEN. Two basic residues, K125 and K128 at the active sitelikely account for the capacity of the P-loop to accommodate the largePIP₃ as a substrate. The cysteine residue at position 124 forms athiophosphate intermediate with the phosphorylated PIP₃ molecule. R130is involved in catalysis of this phosphoester linkage. The His atposition 123 and Glycine at position 127 are also critical for theconformation of the P-loop structure. The trough region defined by theactive site is extended to 8 angstroms in depth and 5×11 angstromopening to the active site. This site will be targeted for molecularmodeling to develop inhibitors specific for the PTEN phosphatase.Comparisons with other phosphatases (PTP and PTP1B) with PIP₃ activitywill facilitate identification of those agents which specificallyinteract with PTEN.

The WPD loop or DHNPPQ motif (residues 92-97; SEQ ID NO: 10) is equallyimportant in catalysis in that mutation of Asp-92 results in a loss ofcatalytic activity. This aspartic acid residue acts as a general acid toprotonate the phenolic oxygen atom of a tyrosyl group for tyrosinephosphatases. These data suggest that the mechanistic action of PTEN issimilar to that of tyrosine phosphatases during hydrolysis of thephosphate ester in PIP₃.

The invariant sequence in the WPD loop will also be used in acombinatorial drug screen based on the electrostatic chargecharacteristics of this region of the PTEN molecule. Functional sidechains of these amino acids will be targeted with organic moleculeswhich mimic or disrupt the WPD interaction with the P-loop and TI-loopresidues. For these screens a PTEN inert organic scaffold will bedeveloped to allow for detection of organic molecules which specificallybind PTEN or PTEN fragments.

The structure of the TI loop in the N terminal domain facilitatesPTEN-mediated dephosphorylation of the PIP₃ molecule by providing anelongated and enlarged catalytic site. This is in contrast to other dualspecificity phosphatases. Residues (164-174) in the T1 loop includeKGVTIPSQRRY; (SEQ ID NO: 17). These residues are 100% conserved. Theserine residue at position 170, R at 173 and Y at 174 in this PTENpeptide are important in maintaining the interaction between the TI loopand the C2 domain and are often mutated in human tumors.

The TI loop of PTEN is in close proximity to the C2 domain and maintainsa rigid interface to promote the open configuration for PIP₃ binding toPTEN. A 100% conserved region in C2 domain, HFWVNTFFI, (SEQ ID NO: 11)will also be used to screen a combinatorial library for organicmolecules which bind to this motif.

Small molecules which have affinity for the C terminal tail of PTEN willalso be screened and characterized. Molecules so identified shoulddecrease degradation of PTEN in cells by interfering withphosphorylation of residues, S380, T382 and T383 within the sequenceRYSDTTDS (SEQ ID NO:16) at the extreme C terminus. Screens will beperformed with recombinant GST PTEN protein comprising the last 50 aminoacids (residues 350-403) of PTEN to identify those agents which haveaffinity for this region of PTEN. This methodology should identifyagents that will antagonize the phosphorylation of PTEN tail and/orinterfere with PDZ binding thereby blocking PTEN interaction with theplasma membrane. Agents which disrupt this PDZ interaction will likelyinterfere with PTEN degradation and hence increase PTEN activity levelsin vivo.

Another suitable target present in the C-terminal domain of PTEN is thePEST domain. This region encodes a site for ubiquitin mediateddegradation of PTEN which, if blocked, should augment PTEN activity bypreventing its degradation. Other sequences present in the C terminus ofPTEN between residues 251-351 include TLTKNDLD-FTKTV (SEQ ID NO:12),GDIKVEF-FTKTV (SEQ ID NO:13), DKANKDKAN-FTKTV (SEQ ID NO:14).

The present invention is not only directed to methods for the rationaldesign and screening of agents having binding affinity to the particularpeptide sequences described above. Several PTEN mutants are disclosedherein which may also be used to advantage to identify molecules whichmodulate PTEN activity. For example, the C124S mutant of PTEN providesan ideal target for the initial screening of therapeutic agents givenits increased affinity for substrates and substrate trapping capacity invivo. Use of this mutant PTEN in the assays of the invention shouldresult in the identification and characterization of phosphoinositol D3analogs that inhibit PTEN activity. Such agents should include organicchemicals with the capacity to disrupt the vicinal sulfhydrylinteraction of C124 with the phosphate group required to form athiol-phosphate intermediate for cleavage of the inositol phosphatebond. Agents which so modulate action of the C124S mutant of PTEN willthen be further assessed in functional phosphatase assays.

Monoclonal antibodies, proteins, protein fragments, peptides andpeptidomimetic analogs of peptides which simulate the binding site forPIP₃ as well as structural homologs of phosphoinositides phosphorylatedin D3 position, or substituted in the D3 position with other negativelycharged functional groups, will be screened for capacity to bind andmodulate PTEN phosphatase activity in vitro. Molecular modeling shouldfacilitate the identification of specific organic molecules withcapacity to bind to the active site of PTEN based on conformation or keyamino acid residues required for catalytic function. A combinatorialchemistry approach will be used to identify molecules with greatestactivity and then iterations of these molecules will be developed forfurther cycles of screening.

The PTEN polypeptide or fragment employed in drug screening assays mayeither be free in solution, affixed to a solid support or within a cell.One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant polynucleotidesexpressing the polypeptide or fragment, preferably in competitivebinding assays. Such cells, either in viable or fixed form, can be usedfor standard binding assays. One may determine, for example, formationof complexes between a PTEN polypeptide or fragment and the agent beingtested, or examine the degree to which the formation of a complexbetween a PTEN polypeptide or fragment and a known substrate isinterfered with by the agent being tested.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to PTEN polypeptides andis described in detail in Geysen, PCT published application WO 84/03564,published on Sep. 13, 1984. Briefly stated, large numbers of different,small peptide test compounds, such as those described above, aresynthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with PTEN polypeptideand washed. Bound PTEN polypeptide is then detected by methods wellknown in the art.

A further technique for drug screening involves the use of hosteukaryotic cell lines or cells (such as described above) which have anonfunctional PTEN gene. These host cell lines or cells are defective atthe PTEN polypeptide level. The host cell lines or cells are grown inthe presence of drug compound. The rate of cellular proliferation andtransformation of the host cells is measured to determine if thecompound is capable of regulating the proliferation and transformationof PTEN defective cells.

Another approach entails the use of phage display libraries engineeredto express fragment of PTEN on the phage surface. Such libraries arethen contacted with a combinatorial chemical library under conditionswherein binding affinity between the PTEN peptide and the components ofthe chemical library may be detected. U.S. Pat. Nos. 6,057,098 and5,965,456 provide methods and apparatus for performing such assays.

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact (e.g., agonists, antagonists, inhibitors) in orderto fashion drugs which are, for example, more active or stable forms ofthe polypeptide, or which, e.g., enhance or interfere with the functionof a polypeptide in vivo. See, e.g., Hodgson, (1991) Bio/Technology9:19-21. In one approach, discussed above, the three-dimensionalstructure of a protein of interest or, for example, of theprotein-substrate complex, is solved by x-ray crystallography, bynuclear magnetic resonance, by computer modeling or most typically, by acombination of approaches. Less often, useful information regarding thestructure of a polypeptide may be gained by modeling based on thestructure of homologous proteins. An example of rational drug design isthe development of HIV protease inhibitors (Erickson et al., (1990)Science 249:527-533). In addition, peptides (e.g., PTEN polypeptide) maybe analyzed by an alanine scan (Wells, (1991) Meth. Enzym. 202:390-411).In this technique, an amino acid residue is replaced by Ala, and itseffect on the peptide's activity is determined. Each of the amino acidresidues of the peptide is analyzed in this manner to determine theimportant regions of the peptide. It is also possible to isolate atarget-specific antibody, selected by a functional assay, and then tosolve its crystal structure. In principle, this approach yields apharmacore upon which subsequent drug design can be based.

It is possible to bypass protein crystallography altogether bygenerating anti-idiotypic antibodies (anti-ids) to a functional,pharmacologically active antibody. As a mirror image of a mirror image,the binding site of the anti-ids would be expected to be an analog ofthe original molecule. The anti-id could then be used to identify andisolate peptides from banks of chemically or biologically produced banksof peptides. Selected peptides would then act as the pharmacore.

Thus, one may design drugs which have, e.g., improved PTEN polypeptideactivity or stability or which act as inhibitors, agonists, antagonists,etc. of PTEN polypeptide activity. By virtue of the availability ofcloned PTEN sequences, sufficient amounts of the PTEN polypeptide may bemade available to perform such analytical studies as x-raycrystallography. In addition, the knowledge of the PTEN protein sequenceprovided herein will guide those employing computer modeling techniquesin place of, or in addition to x-ray crystallography. Suitable peptidetargets for identifying specific PTEN binding and modulating agents areprovided in Table I

TABLE I PTEN peptide motifs used to screen forPTEN agonists and inhibitors Phosphorylation site amino acid residuemotifs: number DLDLTYIYP (22-30) SEQ ID NO: 3 YLVLTL (27-30)SEQ ID NO: 6 YRNNIDD (46-52) SEQ ID NO: 8 KGVTIPSQRRYVYYYSYLL (164-182)SEQ ID NO: 15 YSYL (178-181) SEQ ID NO: 7 YFSPN (336-339) SEQ ID NO: 5RYSDTTDS (378-385) SEQ ID NO: 16 Catalytic Domain motifs (1-185)HCKAGKR(P-loop) (123-130) SEQ ID NO: 9 DHNPPQ (WPD-loop) (92-97)SEQ ID NO: 10 KGVTIPSQRRY (TI-loop) (164-174) SEQ ID NO: 17C2 domain motifs (186-350) HFWVNTFFI (272-280) SEQ ID NO: 11C terminal tail related (351-403) regions of interest TLTKNDLD---FTKTV(319-351) (PEST domain sequences) SEQ ID NO: 12 GDIKVEF---FTKTV(251-351) (PEST domain sequences) SEQ ID NO: 13 DKANKDKAN---FTKTV (PEST)(331-351) SEQ ID NO: 14 RYSDTTDS (pre-PDZ region) (378-385)SEQ ID NO: 16 HTQITKV (PDZ-MAGI-2 (399-403) interaction domain)SEQ ID NO: 18

B. Pharmaceuticals and Peptide Therapies

The elucidation of the role played by PTEN in cellular transformationand angiogenesis facilitates the development of pharmaceuticalcompositions useful for treatment and diagnosis of PTEN associateddisorders. These compositions may comprise, in addition to one of theabove substances, a pharmaceutically acceptable excipient, carrier,buffer, stabilizer or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material may depend on the route of administration, e.g. oral,intravenous, cutaneous or subcutaneous, nasal, intramuscular,intraperitoneal routes.

Whether it is a polypeptide, antibody, peptide, nucleic acid molecule,small molecule or other pharmaceutically useful compound according tothe present invention that is to be given to an individual,administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual.

C. Methods of Gene Therapy

As a further alternative, the nucleic acid encoding the authenticbiologically active PTEN polypeptide could be used in a method of genetherapy, to treat a patient who is unable to synthesize the active“normal” polypeptide or unable to synthesize it at the normal level,thereby providing the effect elicited by wild-type PTEN and suppressingthe occurrence of “abnormal” PTEN associated diseases such as cancer.

Vectors, such as viral vectors have been used in the prior art tointroduce genes into a wide variety of different target cells. Typicallythe vectors are exposed to the target cells so that transformation cantake place in a sufficient proportion of the cells to provide a usefultherapeutic or prophylactic effect from the expression of the desiredpolypeptide. The transfected nucleic acid may be permanentlyincorporated into the genome of each of the targeted cells, providinglong lasting effect, or alternatively the treatment may have to berepeated periodically.

A variety of vectors, both viral vectors and plasmid vectors are knownin the art, see U.S. Pat. No. 5,252,479 and WO 93/07282. In particular,a number of viruses have been used as gene transfer vectors, includingpapovaviruses, such as SV40, vaccinia virus, herpes viruses includingHSV and EBV, and retroviruses. Many gene therapy protocols in the priorart have employed disabled murine retroviruses.

Gene transfer techniques which selectively target the PTEN nucleic acidto malignant tissues are preferred. Examples of this includereceptor-mediated gene transfer, in which the nucleic acid is linked toa protein ligand via polylysine, with the ligand being specific for areceptor present on the surface of the target cells. Microcapsule baseddelivery systems are also available for delivery of nucleic acids totargeted cell types.

The following Examples are provided to describe the invention in furtherdetail. The Examples are intended to illustrate and not to limit theinvention.

Example 1 Orthotopic Brain Tumor Model

To determine whether PTEN exerts control over angiogenesis and/or thegrowth of glial tumors, an orthotopic brain tumor model was developed inwhich PTEN-deficient tumor cells were genetically manipulated in vitroand then stereotactically injected into the frontal cerebral cortex ofnude mice. The U87MG cell line employed in these studies is derived froma patient diagnosed with glioblastoma multiforme, a highly malignant anduniformly fatal brain tumor. This tumor and other human glioblastomasand glioblastoma cell lines contain a mutation in both PTEN alleles(U87MG cells have a homozygous mutation in PTEN resulting in a nullgenotype).

In the orthotopic brain tumor model, 100% of mice implantedintracranially with the parental U87 cells display a highly invasive andangiogenic pattern of brain tumor growth that results in mortalitywithin 25-27 days. In view of these observations, the effect ofreconstituting expression of the PTEN gene in the parental U87MG (U87)cell line was explored.

Stable derivatives of the parental U87 cells were generated followingtransduction with retroviruses encoding cDNA for wild type PTEN orspecific mutants of this phosphatase. In particular, missense mutationsin the PTP signature motif were introduced to ascertain the importanceof the enzymatic activity of PTEN to its tumor suppressor function.Missense mutations included the G129E mutant, which displays a severelyattenuated ability to dephosphorylate inositol phospholipids, butretains normal enzymatic activity for phosphoproteins. The biologicalsignificance of the G129E mutant was underscored by the fact that itspresence has been correlated with Cowden's disease and endometrialcancer. In another missense mutation generated, the R130M mutant, allphosphatase activity has been abrogated (Myers et al PNAS 1997, Funariet al PNAS 1997).

Tumor cells were characterized biochemically for levels of activated AKT(phospho-S473-AKT), growth in vitro and PTEN expression (FIG. 1).Anti-PTEN blots confirmed that parental U87 cells did not express PTENand that following reconstitution of PTEN expression, U87 cellsexpressed significant and comparable amounts of the wild type or mutantphosphatase protein. Expression of wild type PTEN, at levels similar tothose observed in a mouse brain lysate, suppressed the activated stateof AKT observed in PTEN-deficient U87 cells (FIG. 1A, lanes 3 & 5).Following expression of the R130M and G129E mutant forms of PTEN, thelevels of phospho-AKT were similar to those observed in the parental U87cells (FIG. 1A, lanes 1, 2 & 4), suggesting that the lipid phosphataseactivity of PTEN was essential for the effects on the PIP3-dependentactivation of AKT. Interestingly, the growth of the differentPTEN-expressing U87 cell lines in vitro was similar in 2, 5 and 10%fetal bovine serum (data not shown and FIG. 1B). Therefore, we comparedthese cell lines further in our in vivo models.

Athymic nude mice were implanted subcutaneously and by intracranialinjection. Production of subcutaneous tumors facilitates monitoring oftumor size and performance of direct biochemical analysis of tumortissue for the examination PTEN expression and levels of AKT activationwithout significant contamination from other tissues. Tumor tissueblocks were processed for H & E staining, which confirmed that >95% ofthe tissue examined comprised tumor cells free of dermal or subdermaltissue. The levels of PTEN in tumor tissue and numerous normal tissueswithin the athymic nude mouse were compared. Using anti-PTEN antisera,the expression of PTEN was detected in all tissues, with the exceptionof skeletal and heart muscle (data not shown). No PTEN was detected inparental U87-derived tumor tissue (FIG. 2C, lane 4). These resultsdemonstrate that the tumor tissue sampled contained predominantly tumorcell-derived proteins. As observed in the cell lines grown in vitro,subcutaneous tumors derived from U87 cells reconstituted with mutant orwild type PTEN display similar levels of PTEN expression (FIG. 2C, lanes1, 2, 3 & 5). Phospho-AKT activity was higher in PTEN-null U87 cells andU87 cells reconstituted with R13QM and to a lesser extent in U87 cellsexpressing the G129E mutant (FIG. 2C, lane 1) as compared to the wildtype PTEN transduced cells (FIG. 2C, compare lanes 1, 2 & 4 to lanes 3 &5). The pattern of phosphorylated AKT was similar when the different U87mutant expressing cell lines were assayed in vitro or in vivo (compareFIG. 1A to 2C). Despite the similar in vitro growth rate, there was adramatic difference in the growth of tumors derived from parental U87cells compared to cells reconstituted with wild type PTEN (FIGS. 2A &B). The average volume of U87-derived tumors on day 25 afterimplantation was 848±203 mm3, compared to 91±27 mm3 for tumors derivedfrom PTEN-reconstituted cells (n=5, p<0.0001). Reconstitution withcatalytically dead mutants of PTEN significantly reduced the rate ofgrowth in vivo without a demonstrable effect on angiogenesis (FIGS. 2Aand 3C). Others have observed effects of catalytically dead PTENexpression on cell invasion, suggesting a function for other regions ofthe PTEN molecule in cellular functions. Interestingly, in vivo BrdUlabeling of tumor cells revealed no significant difference in number ofcells in S phase (72±6 BrdU positive cells per field in parental U87MGtumor mass versus 68.5±3 in WT PTEN reconstituted tumors). These datademonstrated that the loss of PTEN-mediated inositol phospholipidphosphatase activity was a critical component of deregulated tumorgrowth. Notably, the G129E mutant (which lacks inositol phospholipidphosphatase activity) was equivalent to the R130M mutant (which lackedinositol phospholipid and phosphoprotein activity) with respect to bothtumor growth and the proliferative rate of these tumors in vivo.

To assess the effect of PTEN on angiogenesis, parental U87 cells werecompared to cells reconstituted with wild type or mutant PTEN. Cryostatsections from subcutaneous tumors for were stained for CD31 (PECAM), anendothelial marker used to measure the microvessel density of thesetumors. Microvessel density was assessed from multiple digitized imagesof CD31-stained tumor tissue at 100×magnification (3 fields wereevaluated per tumor) and counted blindly for the number of CD31 positivemicrovessels per unit surface area as described (Weidner et al 1991 NEngl J Med 324:1-8). Reconstitution of PTEN expression in U87 cellsdramatically suppressed the angiogenic response in vivo (FIGS. 3A & B).Quantitation of microvessel density in tumors derived from parental U87cells (77±13) and U87 cells expressing wild type PTEN (38±7) revealed an˜50% suppression of angiogenesis (FIG. 3C) (n=5, p<0.001). Themicrovessel density of tumors derived from U87 cells reconstituted withcatalytically impaired PTEN (R130M, 84±15 or G129E, 69±16) were notsignificantly different (p>0.05) from the parental U87 cell line (FIG.3C). Similar results were obtained from an analysis of microvesseldensity of intracranial tumors. The levels of phospho-AKT detectedwithin the tumor mass in vivo demonstrated a mechanistic link betweenthe loss of the inositol lipid phosphatase function of PTEN, thephosphorylation status AKT and the angiogenic phenotype within thetumor.

Recent in vitro data suggested a link between PTEN and downstreamtargets including AKT, HIF1α and VEGF in the potential control ofangiogenesis (Zundel et al., Genes Dev 2000, Zhong et al., Cancer Res2000). We used the RNase protection assay (RPA) to examine the effect ofPTEN on thrombospondin 1 (TSP-1) expression. RPA was performed with aTSP-1 specific probe in U87MG cells constitutively expressing wild typePTEN or G129R PTEN (FIG. 3D). The data demonstrate that wild type butnot mutant PTEN expression induces TSP-1 in U87 cells. To confirm theseresults, Western blot analysis was performed to assess TSP-1 expressionin a retroviral-based ecdysone-inducible PTEN expression system (No etal PNAS 1996). Inducible and dose-dependent expression of PTEN wasconfirmed in U87 cells. The induced expression of wild type PTEN, butnot G129R PTEN, resulted in augmentation of thrombospondin 1 expression(FIG. 3E) and suppression of AKT activation as demonstrated by decreasedphospho-AKT levels without an accompanying decrease in total AKT (datanot shown). The induced expression of mutant G129R had no effect onphospho-AKT levels. The data therefore demonstrate that PTEN positivelymodulated the expression of thrombospondin 1, a negative regulator ofangiogenesis (FIGS. 3D and E) (Sheibani and Frazier, (1999) Histol.Histopathol. 14:285-294, Hsu et al., (1996) Cancer Res. 56:5684-5691).These data suggested that PTEN has a pivotal role in angiogenesismediated, in part, by the induction of TSP-1.

Vascular endothelial growth factor (VEGF) is a known positive regulatorof angiogenesis (Jiang et al. (2000) Proc Natl Acad Sci USA, 97(4),1749-53; Mazure et al. (1997) Blood, 90(9), 3322-31; Mazure et al.(1996) Cancer Res, 56(15), 3436-40; Plate et al. (1994) Int J Cancer,59(4), 520-9; Plate et al. (1992) Nature, 359(6398), 845-8). Thecapacity of tumor tissue to produce VEGF was determined for U87MGparental cells null for PTEN versus U87 cells reconstituted with wildtype PTEN or mutants of PTEN (G129E or R130M). Tumor tissue obtainedfrom subcutaneously implanted tumor cells was subjected to cryostatsectioning and multiple sections through the tumor tissue were pooledfor biochemical Western blot analysis using anti-VEGF antibody (SantaCruz, SC-507). Cell lysates were assayed for protein concentration byBradford method. Equivalent amounts of total protein were resolved bySDS PAGE followed by immunoblot for VEGF protein.

The results demonstrate that the reconstitution of wild type PTEN butnot catalytically dead PTEN markedly suppresses the production of VEGFby U87MG tumors in vivo. No VEGF was detected in tumors reconstitutedwith the wild type PTEN (FIG. 4, lanes 3 and 4). An intermediate levelof suppression is noted in tumors reconstituted with the G129E mutant ofPTEN (FIG. 4, lane 1), a mutant which has lost its capacity todephosphorylate PIP₃ and not protein substrates. These data show thefirst evidence that PTEN suppresses VEGF, a proangiogenic growth factorin vivo. The data implicate PTEN and therefore, the PI-3 kinase cascadein the coordinate regulation of the “angiogenic switch” mechanism whichcontrols angiogenesis under normal physiologic conditions. It is thiscontrol that is lost during tumor progression.

Brain tumor-induced angiogenic responses are known to occur in thecontext of brain specific stromal and extracellular matrix interactions.To determine whether the expression of PTEN affected the survival ofmice in an orthotopic brain tumor model, U87 cells expressing eitherwild type or mutant forms of PTEN were implanted under stereotacticcontrol into the right frontal lobe of nude mice (FIG. 5A-D, see arrowfor site of implantation). The results demonstrated that reconstitutionof wild type PTEN in U87 cells suppressed the malignant potential ofthese cells in an orthotopic animal model. Thus, there was 90% survivalat 40 days in animals implanted with the wild type PTEN-reconstitutedU87 cells compared to 100% mortality of mice implanted with the parentalcells at 27 days (FIG. 5E) (n=15, p<0.0001). PTEN reconstituted tumorcells grew more slowly when implanted in the frontal lobe (FIG. 5,compare A & B) and remained circumscribed to that area of brain (datanot shown). U87 cells reconstituted with PTEN mutants, ablated foreither inositol lipid phosphatase activity (G129E) or all phosphataseactivity (R130M), displayed a phenotype similar to the PTEN-negative,parental U87 cells (FIG. 5C). Animals with tumors derived from U87 cellsreconstituted with PTEN-G129E displayed slightly prolonged survival (50%at day 30) compared to those implanted with parental U87 cells; all ofthese animals were dead, however, by day 40. These data implicate theinositol lipid phosphatase activity of PTEN is required for controlledangiogenesis (FIG. 3) and its loss is correlated with the development ofa highly malignant glioma (FIG. 5C) following implantation of U87 tumorcells in mice.

Example II PTEN Reconstitution Reduces Metastatic Potential of BrainTumor Cells Introduced Via the Carotid Artery and Negatively RegulatesProangiogenic Factors

The following methods are provided to facilitate the practice of ExampleII.

As described in Example I, wild type PTEN or mutant PTEN (G129E, R130M)cDNAs were subcloned into the pBabe-puro retroviral expression vector.Stable clones of U87MG cells were established under puromycin selection(2 ug/ml) (Myers et al. 1998). A panel of antibodies were obtained whichare immunospecific for PTEN (Myers et al. 1998), AKT, phospho-AKT (NewEngland Biolabs, #9270), TIMP-3, MMP-9, and MMP-2 (Sigma).

To evaluate the role PTEN plays in regulating the expression of factorsthat promote angiogenesis and invasiveness of glial tumors, an in vitrogelatin zymography assay was performed. Gelatin zymography specificallydetects the presence of members of this matrix metalloproteinase (MMP)family of proteins, which degrade the ECM, thereby promotingangiogenesis and metastasis. The assay was performed using either 1)lysates derived from tumor tissue or 2) conditioned media in which thetumor cells or tissue were maintained. Protein samples for analysis weregenerated from isolated subcutaneous tumors by dissolving the tumortissue in a detergent lysis buffer [50 mM Tris-Cl, (pH 8.0), 150 mMNaCl, 0.05% NP-40, 100 mM NaF, 1 mM EDTA, 1 mM EGTA, 0.08 mM PMSF, 0.01mg/ml leupeptin, 0.01 mg/ml aprotinin, 1 μmg/ml pepstatin A]. Proteinsamples for analysis were also generated from tumor cell conditionedmedia concentrated by centrifugation through Microcon® YM-10 centrifugalfilter devices. The protein concentration of samples composed of eithertumor cell lysate or concentrated, conditioned media derived from thevarious tumor cell cultures was determined by standard protein assay(Bio-Rad, Hercules, Calif.). Protein samples were then normalized forequal protein concentrations.

Protein samples (10 μg) were subjected to substrate gel electrophoresiswith modifications. Briefly, protein samples normalized for proteinconcentration were applied, under non-reducing conditions, to 10%polyacrylamide slab gels impregnated with 1 mg/ml gelatin (DIFCO). Afterelectrophoresis, the gel was washed at room temperature for 30 minutesin washing buffer [50 mM Tris-Cl (pH 7.5), 5mM CaCl₂, 1 mM ZnCl₂, 2.5%Triton X-100] and then incubated overnight at 37° C., with gentleagitation, in washing buffer containing 1% Triton X-100. The gels werestained with a solution of 0.1% Coomassie brilliant Blue R-250. Clearzones in the gel are indicative of the presence of gelatinolyticactivity contained in a given protein sample. The gelatinolytic activitywas quantitated by densitometric scanning and analysis

To further evaluate the role PTEN plays in regulating the expression offactors that promote angiogenesis and invasiveness of glial tumors,Matrigel® invasion assays were also performed. Matrigel® invasion assaysare a standard procedure used to characterize the metastatic potentialof cells, based on the ability of such cells to degrade the Matrigel®extracellular matrix (ECM). Matrigel® is a commercially available mix ofbasement membrane components, generated from an EFS sarcoma, whichincludes basic components of the basement membrane such as collagens,laminin, and proteoglycans, as well as matrix degrading enzymes, theirinhibitors, and growth factors. Invasion of tumor cells into Matrigel®has been used to characterize involvement of matrix-degrading enzymeswhich play important roles in tumor progression and metastasis (Benelliand Albini, 1999).

The assay was performed as follows: Matrigel® (Becton-Dickinson) wasthawed overnight at 4° C. on ice and diluted to 1 mg/ml in serum-freeDMEM. 50 μl of the diluted Matrigel® was added to the upper chambers ofa 24-well Transwell® plate (0.8 μm pore size, Costar). The upperchambers were then incubated at 37° C. for 6 hours to facilitatesolidification of the gelatinous matrix. U87 glioma cells were harvestedfrom tissue culture flasks by 0.04% Trypsin/EDTA and washed three timeswith serum-free DMEM to remove trace amounts of sera. Cells wereresuspended in serum-free DMEM at a density of 5×10⁵ cells/ml, 200 μl ofwhich was added to upper chambers coated with solidified Matrigel®. Thelower chambers of the Transwell® plate were filled with 600 μl of DMEMcontaining 5 μg/ml fibronectin, which served as a soluble attractant andadhesive substrate. Transwell® plates were incubated at 37° C. for 36hours to facilitate migration of U87 cells from the upper chambers intowhich they were seeded to the lower chambers. Following this incubationtime, the upper chambers were removed and stained with 0.1% crystalviolet solution to visualize the cells. Cells that failed to migrate tothe underside of the porous membrane that separates the top and bottomchambers of the Transwell® were removed by scraping the topside of themembrane with a cotton-tipped swab. Cells that had successfully migratedthrough the Matrigel® to the underside of the porous membrane werecharacterized as invasive cells and counted under 100× magnification.

An in vivo study of experimental metastasis following orthotopicintroduction U87 glioma cells into the nude mouse brain was alsoundertaken. U87MG cells were introduced into the circulatory system of amouse via the injection of cells into the intracarotid artery. Eachmouse was anesthetized by intraperitoneal injection of Nembutal andrestrained on a cork board equipped with fixed rubber bands that wereused to wrap around the teeth of the upper jaw, thereby immobilizing thehead. Under a dissecting microscope, the hair over the trachea (if amouse species having hair was used) was shaved, the neck was preparedfor surgery with betadine, and the skin cut by a mediolateral incision.After blunt dissection, the trachea was exposed and the musclesseparated to expose the right common carotid artery, which was thenseparated from the vagal nerve. Further dissection was then performed toreveal the internal and external carotid arteries. The common carotidartery was prepared for injection distal to the point of divisionbetween the internal and external carotid arteries. Briefly, a ligatureof 5-0 silk suture was placed in the distal portion of the commoncarotid artery and a second ligature was positioned and tied looselyproximal to the injection site of the internal carotid artery. A sterilecotton tip applicator was inserted under the artery just distal to theinjection site to elevate the artery. This procedure controlled bleedingfrom the carotid artery by regurgitation from distal vessels. The arterywas nicked with a pair of microscissors, and a plastic cannula (<30gauge) was inserted into the lumen and threaded forward into theinternal carotid artery. Wild type cells or cells expressing the variousmutants of PTEN, G129R, G129E, R130M, (1×10⁶ in 10 μl) resuspended inPBS were injected slowly into the artery, after which the cannula wasremoved. The second ligature was then tightened and the incision in theskin sealed with surgical clips.

Following completion of the surgery, mice were placed in clean cagesequipped with heating sources to maintain their body temperature. Themice were then monitored until they recovered from the effects of theanesthesia and returned to the care of the animal facility. Deleterioussystemic effects associated with brain lesions, such as cachexia,listlessness, and protrusion of the right bulb were monitored. Mice weresacrificed when moribund. Brains were removed and fixed in 10%formaldehyde for H&E staining and in OCT for frozen sections.

Cell lysates obtained from U87 cells grown in tissue culture or frommultiple cryostat sections of U87MG subcutaneous tumor tissues were alsorun on gels and analyzed by Western blotting. A Bradford assay wasperformed to determine protein concentration of each lysate. Equivalentamounts of protein were resolved by SDS PAGE and transferred tonitrocellulose. Membranes were probed with antisera specific for PTEN,AKT, phospho-AKT or TIMP-3. The RNAase protection assay (RPA) wasperformed using a RPA III kit from (Ambion) according to the manufacturespecifications. Briefly, 20 μg of total RNA was precipitated andresuspended in 10 μl of hybridization buffer containing specificradioactive probe. The RNA was then heated to 95° C. for 10 min andhybridized for 16 hours at 42° C. 150 pl of this mixture was treatedwith 1:100 dilution of RNAase in RNAase buffer for 30 minutes. TheRNAase was inactivated and the RNA was reprecipitated and resolved on 5%acrylamide gel. RNA probes were synthesized using MAXI Script utilizingPCR templates and T7 polymerase. The GAPDH probe was provided in the kitas an internal control. The TIMP-3 probe represented a 590 nucleotidesequence located in the 3′ UTR of the TIMP-3 sequence. All probes weresequenced.

Microvessel density (MVD) was determined for each brain tumor asdescribed in Example I by CD31 staining, performed on cryostat sections(7 μm), fixed in acetone, blocked in 1% goat serum and stained withanti-CD31 antibody (Pharmingen, #01951D). Antibody staining wasvisualized with peroxidase-conjugated anti-mouse antibody and counterstained with hematoxylin. A negative control was performed on each tumortissue stained with mouse IgG. Two sections from each tumor were scannedunder low power magnification (40×) to identify areas of highest CD31positive vessel density (Weidner et al. 1991), followed by digitizationof 5 fields from this area. The digitized images representing one 100×field were counted for the number of CD31 positive vascular elements.Data was collected independently by two researchers in a blind study.The average number of microvessels per digitized 100× field wasdetermined for 5 tumors per experimental group and analyzed by Student'st-test.

The data obtained thus far indicated that the anti-angiogenic activityof PTEN can be correlated matrix remodeling and degradation. We used theRNAase protection assay (RPA) to confirm the effect of PTEN on TIMP-3expression. RPA was performed with a TIMP-3 specific probe in U87MGcells constitutively expressing wild type PTEN or G129R PTEN. The datademonstrated that wild type, but not mutant PTEN expression inducedTIMP-3 expression in U87 cells. Thus, PTEN positively modulates theexpression of TIMP-3, a negative regulator of matrix metalloproteinases(FIG. 6). These data reveal a novel mechanism through which PTENregulates angiogenesis and extracellular matrix remodeling via theinduction of TIMP-3 and suppression of matrix metalloproteinase levels.To confirm this observation, matrix metalloproteinase activity in PTENnull and wild type PTEN reconstituted tumors (FIG. 7), was examined byperforming reverse zymography for collagenolytic activity. Consistentwith the finding that PTEN induced TIMP-3 expression in U87 tumor cells,it was apparent that wild type PTEN reconstitution suppressed MMP-9activity with no effect upon MMP-2 activity (FIG. 7) (Oh et al. 1999;Rao et al. 1994).

To determine the role PTEN play is tumor cell invasion, PTEN deficientand PTEN reconstituted glioma cells were evaluated for their capacity toinvade and migrate through a matrigel coated membrane. PTENreconstitution was observed to completely abrogate invasion of U87 tumorcells through matrigel (FIG. 8). Adhesion and migration on matrigelremained intact in PTEN reconstituted U87MG cells (data not shown).Importantly, the data provided the first direct evidence that PTENcontrols matrix degradation and the invasive behavior of tumor cells invivo, and suggest that PTEN as well as PI-3 kinase inhibitors willsuppress tumor invasion and the metastatic phenotype in vivo.

The data described above clearly demonstrate that mutations in the PTENtumor suppressor in U87 glioblastoma cells resulted in the loss ofnormal physiologic control of matrix remodeling and invasion.Furthermore, analysis of the data revealed that PTEN exerts its effectat the level of TIMP-3 expression and control of MMP-9 matrixmetalloproteinase activity. The results, therefore, implicate PI-3kinase and other downstream targets of PTEN such as AKT, in the controlof metastasis and invasion of tumor cells. The U87 brain tumor modeltherefore provides an ideal assay system to identify agents thatnegatively and/or positively regulate the activities of PTEN or othersignaling molecules in the pathway such as VEGF, or bFGF, etc. whichcontrol matrix degradation in vivo. Finally, the results provided thefirst direct evidence that PTEN controls the capacity of a tumor todegrade the extracellular matrix in vivo. As mentioned previously, PTENpossesses lipid phosphatase activity which preferentiallydephosphorylates phosphoinositides at the D3 position of the inositolring. To date, PTEN is only one of two enzymes reported to have thisactivity. Reconstitution of PTEN in tumor cells that carry mutations inthe PTEN gene, have established that this phosphatase regulates the PI-3kinase-dependent activation of AKT, a major player in cell survival.This observation indicates that PTEN may function as a direct antagonistof PI-3 kinase and PIP-3 dependent signaling. Accordingly, P1-3 kinaseinhibitors, such as LY294002, should be efficacious in blocking matrixdegradation, invasion and metastasis of tumors in vivo. Finally, sincethese pathways are also important in wound healing, the data provide thefirst mechanistic link between P1-3 kinase signaling pathways and PTENsignaling pathways in the control of wound healing and wound associatedangiogenesis.

Example III Inflammatory Signaling Downstream of Fe Receptor Activationis Modulated by Agents that Alter Tyrosine Kinase and PhosphataseActivity

Fc gamma receptor mediated phagocytosis is a model for immunoreceptor(ITAM) signaling and involves the activation of protein tyrosine kinasesand protein tyrosine phosphatases. Relatively little is known of role oflipid phosphatases in control of ITAM signaling and inflammation. Weused phagocytic J774A.1 cells and a heterologous COS7 cell system toexamine the roles played by Src family of protein tyrosine kinases, Sykand PI-3 kinase and protein tyrosine phosphatase, PTEN in signaltransduction pathway leading to Fcγ receptor mediated phagocytosis.Heterologous expression of dominant negative Syk in J774A.1 cellssignificantly inhibited phagocytosis of sensitized sRBCs andpreincubation of the cells with the Src specific protein tyrosine kinaseinhibitor, PP1, and PI-3 kinase inhibitor, Wortmannin, was shown toinhibit this response. Stimulation of J774A.1 cells with sensitizedsRBCs induced tyrosine phosphorylation of Cbl, which was inhibitedsignificantly by PP1 and to some extent by heterologous expression ofdominant negative Syk implicating Src and syk in the phosphorylation ofCbl in phagocytic signal transduction. The heterologous overexpressionof PTEN completely abrogated the phagocytosis of IgG sensitized sheepred blood cells (sRBCs) compared to catalytically inactive mutant ofPTEN. These data provide the first evidence that PTEN, a tyrosinephosphatase, is involved in the regulation of Fcγ receptor mediatedphagocytosis and the regulation of immunoreceptor tyrosine basedactivation motif (ITAM) based signaling events in hematopoietic cells.

Overexpression of a catalytically dead PTEN phosphatase resulted inaugmented phosphorylation of AKT and augmentation of phagocytosis andITAM signaling. These results show that activating signals provided bySrc family kinases, Syk and P1-3 kinase are opposed by inhibitorysignals through PTEN in the control of Fcγ receptor mediatedphagocytosis. Thus, PTEN agonists and P1-3 kinase inhibitors shouldprovide potent anti-inflammatory and immunosuppressive agents to controldownstream signaling events mediated by ITAM linked receptors found inhematopoeitic cells (e.g., T cell receptor, B cell receptor, Fcreceptors and collagen receptor VI). Such agents should also exertpotent control over the immune responses mediated through T cells, Bcells, myeloid cells including macrophages, neutrophils, dendriticcells, monocytes, mast cells and platelets by preventing theiractivation and subsequent modulation of unwanted immune reactivity andplatelet aggregation which contribute to a number of human diseases.

The following methods and materials are provided to facilitate thepractice of Example III.

Anti-Cbl antibody was obtained from Santa Cruz Biotechnology, SantaCruz, Calif. Anti-Syk antibody was provided by Dr. Tamara Hurley (TheSalk Institute, San Diego, Calif.) and anti-PTEN antibody was generatedby immunizing rabbits with an N-terminal peptide of PTEN.

J774A.1, a macrophage-like cell line, and Cos 7 cells were obtained fromthe ATCC. Both cell lines were maintained in DMEM supplemented with 10%fetal calf serum (FCS). Recombinant vaccinia virus vectors were providedby Dr. Bernard Moss (National Institutes of Health, Bethesda, Md.). Thedominant negative Syk vaccinia construct (encoding the amino terminalresidues 1-255 of Syk) which also encodes beta-galactosidase wasprovided by Dr A. Scharenberg (Sharenberg et al., (1995) Embo J.14:3385). Recombinant vaccinia viruses containing PTEN and dominantnegative Syk were prepared as described below.

Briefly, recombinant vaccinia viruses were propagated in 149B cellsgrown in RPMI medium containing 10% FCS. A confluent culture of cellswas infected with recombinant vaccinia virus at a concentration of 0.5pfu/cell for 48 h. The cells were scraped from the plastic in the samemedium, centrifuged to generate a cell pellet, and resuspended in 5 mlof 10 mM Tris-HCl (pH 9). The cells were lysed by three cycles offreezing in liquid nitrogen and thawing at 37° C., after which thevolume of the cell lysate was adjusted to 20 ml with 10 mM Tris-HCl (pH9) in preparation for mechanical lysis step provided by forty strokes ina homogenizer. Nuclei and cell debris were separated from the celllysate by centrifugation at 1,000 rpm for 5 min. The cell lysatecontaining the recombinant vaccinia virus was then subjected tosonication for 1 min with 50% output and a 30% duty cycle. The celllysate was loaded on a cushion of 36% sucrose solution and centrifugedat 13,000 rpm for 80 min 4° C. in an ultracentrifuge (Beckman, PaloAlto, Calif.) using a SW.28 rotor. Viral pellets obtained wereresuspended in 1 ml of 10 mM Tris-HCl (pH 9) and loaded onto a sucrosegradient composed of 6.6 ml each of 40%, 36%, 32%, 28% and 24% ofsucrose solutions made in 10 mM Tris-HCl (pH 9) to be centrifuged at12,500 rpm for 50 min at 4° C. in an ultracentrifuge using a SW.28rotor. A bluish white ring containing purified virus was collected anddiluted with 10 mM Tris-HCl (pH 9) prior to a final centrifugation at13,000 rpm for 60 min at 4° C. in an ultracentrifuge using a SW.28 rotorto pellet the virus. Purified recombinant vaccinia virus thus obtainedwas suspended in 10 mM Tris-HCl (pH 9) and titered as followed. Analiquot was used for generating serial dilutions of the concentratedviral suspension. The serial dilutions were used to infect a confluentlawn of 149B cells grown in 35 mm wells for 2 h at 37° C. in 1 ml ofRPMI medium containing 10% FCS. The medium was then replaced with 3 mlof fresh RPMI medium containing 10% FCS. The medium was discarded after24 hours incubation and viral plaques were visualized by staining withcrystal violet to titer the virus.

A DNA construct encoding the catalytically dead trap mutant of PTEN,C124S, was kindly provided by Nicholas Tonks (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). This mutant The PTEN insert wasamplified by PCR using the following 5′ and 3′ primers:5′GG-GTC-CAC-ATG-ACA-GCC-ATC-ATC-AAA-GAG'3′ forward primer (SEQ ID NO:19) and 5′-GG-TCT-AGA-TCA-GAC-TTT-TGT-AAT-TTG-TGA′3′ reverse primer (SEQID NO: 20), respectively. The amplified product was subcloned into thePCR2.1 vector for propagation using the TA cloning kit (Invitrogen, SanDiego, Calif.), and the PTEN insert was cleaved from the vector bydigestion with SmaI and SalI and subsequently ligated into a linearizedrecombinant vaccinia vector pSC65 to generate pSC65-PTEN. The constructC2pSC65 was used to make a recombinant vaccinia virus using thepackaging cell line CV1 and the wild type vaccinia virus. Recombinantvirus was isolated from wild type virus by single plaque purification,and then amplified, purified, and titered as described above.

J774A.1 phagocytic cells were plated at 2×10⁵ cells per well in a twelvewell plate (Costar, Corning, N.Y.) and cultured overnight. Cells wereinfected with recombinant vaccinia virus pSC65 or pSC65-PTEN at adensity of 2 pfu/cell for 4 h at 37° C. in 5% CO₂. The media was changedafter 4 hours and the cells were incubated with sheep red blood cellscoated with IgG at a subagglutinating concentration. The target toeffector ratio was 100:1. The cells were harvested after 2 hours, andcytospins were prepared, fixed, and stained with Wright Giemsa stain(Dade, AG, Switzerland). Stained slides were evaluated microscopicallyfor rosette formation. The remaining uningested sRBCs were subjected tolysis by osmotic shock in water. The cells were suspended in DMEM mediumcontaining 20% FCS. The cells were spun down on glass slide and fixedand stained by Wright Giemsa stain. A minimum of one hundred and fiftycells were counted for each slide and the phagocytic index wascalculated as follows: Phagocytic Index (PI)=% of phagocyticcells×average number of sRBCs engulfed by each cell.

In the drug inhibition studies, the cells were subjected to treatmentwith an indicated inhibitor at different concentrations along with anappropriate DMSO control for 1 hour in DMEM with 10% FCS before carryingout the phagocytic assay.

Assays to detect β-galactosidase activity were performed as follows:cells (1×10⁵) were suspended in 400 μl of DMEM containing 10% FCS towhich 50 μl of 1% X-gal (Sigma, St. Louis, Mo.) was added. Subsequentincubation at 37° C. facilitates β-galactosidase activity which isindicated by acquisition of a blue color by the reaction mixture. Thereaction mixture was diluted 1:10 and the optical density was measuredat 595 nm in a spectrophotometer (Molecular Devices, Menlo Park,Calif.).

J774A.1 cells and COS7 cells (2×10⁵) infected with recombinant virusesexpressing either dominant negative Syk or PTEN were lysed in 50 μl ofsample buffer. The lysates were resolved by SDS-PAGE, transferred to asolid matrix support, and probed to assess protein expression withspecific antibodies.

J774A.1 cells were infected with recombinant vaccinia virus at theconcentration of 2 pfu/ml for 4 hours. The infected cells were thenpelleted by centrifugation, and resuspended at a concentration of 2×10⁶cells per ml in DMEM to be stimulated with IgG coated sRBCs at 37° C.for 5 min. The samples were pelleted at 500×g in a refrigeratedcentrifuge and the resultant cell pellet was lysed as described earlierand analyzed following immunoprecipitation with specific antibodies.

Dominant Negative Syk Inhibits Phagocytosis

In order to investigate the role played by the nonreceptor tyrosinekinase Syk in IgG mediated phagocytosis, a dominant negative mutant formof Syk was expressed in J774A.1 cells utilizing recombinant vacciniavirus as a means of transmission. This Syk mutant encodes a truncatedform of Syk which comprises the tandem SH2 domains, but excludes thecatalytic domain. As a result of this mutation, the protein behaves as adominant negative mutant by binding to the ITAMs of the FcγR subunit,thereby blocking the interaction of the endogenous catalytically activeSyk with these sites. The results demonstrated that the expression ofdominant negative Syk in J774A.1 cells inhibited phagocytosis of IgGcoated sRBCs (FIG. 9A). In contrast, J774A.1 cells infected with emptyvector recombinant vaccinia virus, as a control, engulfed sensitizedsRBCs normally. The expression levels of dominant negative Syk ininfected cells was evaluated by Western analysis using antibodiesspecific for Syk (FIG. 9B, lane 3). To ensure that the inhibitory effecton phagocytosis was specific to the expression of dominant negative Syk,and not a consequence of differential levels of viral infection,infected J774A.1 cells were assessed for viral load. Since the pSC65plasmid, into which the dominant negative Syk was cloned, also containedthe gene encoding β-galactosidase, this enzyme was used as an internalcontrol for viral expression levels. Briefly, β-galactosidase activitycan be quantified colorimetrically as indicated by the appearance of acolored product following cleavage of the substrate β-gal. In everyexperiment, the levels of recombinant viral load were equivalent tothose of control cells (infected with empty vector recombinant vacciniavirus) and experimental cells (infected with vaccinia virus containingdominant negative Syk; data not shown).

As an additional control to evaluate the specificity of the inhibitionobserved after expression of dominant negative Syk, the capacity ofJ774A.1 cells to form rosettes via the FcγR was examined followinginfection with different viruses. Cells infected with empty vectorrecombinant vaccinia virus and cells infected with vaccinia viruscontaining dominant negative Syk displayed 100% rosette formation within1 minute of addition of sensitized sRBCs; these data indicated thatneither the cell surface expression level of FcγRs nor their bindingcapacity for sRBC targets was affected by infection with recombinantvaccinia virus alone (data not shown). Notably, rosette formation andphagocytosis did not occur in the absence of sensitizing antibodyagainst sRBCs, thereby further underscoring the specificity of theseresponses. These data using dominant negative Syk were consistent withother data in the literature, including those derived from Syk knockoutmice (Crowley, M., et al., (1997) J. Exp. Med. 186:1027), which stronglysupport a role for Syk in propagating signals required for IgG mediatedphagocytosis in the J774 system.

Src and PI-3 Kinase are Required for Phagocytosis of IgG Coated sRBCs byJ774A.1 Cells

Recent evidence from Hck,/Lyn/Fgr knockout mice suggests that members ofthe Src family of nonreceptor protein tyrosine kinases function upstreamof Syk and PI-3 kinase in ITAM signaling (Crowley et al., 1997, supra).To examine the role of Src in FcγR-mediated phagocytosis, J774 cellswere treated with different concentrations of PP1 (1, 5, or 10 μM;Calbiochem, La Jolla, California), a Src family tyrosine kinaseinhibitor, wortmannin(1 or 5 μg/ml), an inhibitor of PI-3 kinase(LY294002), or DMSO as a control. Briefly cells were treated with theabove reagents for 1 hour in DMEM supplemented with 10% FCS and thensensitized sRBCs were added at a target to effector ratio of 100:1. Asshown by FIG. 10A, PP1 inhibited phagocytosis in a dose dependent mannerand completely abrogated phagocytosis at a 10 μM concentration. As shownin FIG. 10B, 5 μg/ml wortmannin also mediated significant inhibition ofphagocytosis. These observations implicate the Src kinase family andPI-3 kinase in IgG-mediated phagocytosis of sRBCs by J774A.1 cells.

Effect of Dominant Negative Syk and Src Inhibitor, PP1, on TyrosinePhosphorylation of Cbl in Response to Stimulation with Sensitised sRBCs

It is well known that Fcγ receptor crosslinking induces the tyrosinephosphorylation of the adapter protein, Cbl (Park, R. K., et al., (1996)J. Immumology 160:5018). To determine if phagocytic signaling eventslead to the phosphorylation of Cbl, the degree of Cbl phosphorylationwas assessed before and after induction of phagocytosis. To investigatethe role of specific kinases in this phosphorylation event, dominantnegative Syk and the Src family kinase inhibitor PP1 were utilized toinhibit the activity of these enzymes. The results demonstrated that Cblwas phosphorylated on tyrosine residues following induction ofphagocytosis and this phosphorylation event was abrogated by PP1 (FIGS.11A and 11B, compare lanes 2-3 to 5-6). This effect was dose dependent(data not shown), as was the effect of PP1 on inhibition of Fcγreceptor-mediated phagocytosis (FIG. 11A). Interestingly, dominantnegative Syk inhibited Cbl tyrosine phosphorylation to a lesser extentbut completely abrogated the phagocytic response. Interestingly, bothPP1 and dominant negative Syk suppressed the basal tyrosinephosphorylation levels of Cbl in vivo. These data suggested that thecatalytic activity of the Src family kinases and the capacity of Syk todock with the ITAM receptor were both required for Cbl phosphorylationin response to phagocytic stimuli and that these two events wererequired for phagocytosis. The dominant negative Syk would not beexpected to alter the upstream activity of Src family kinases and henceSrc mediated phosphorylation of Cbl was not altered to the same extent.The data provided support for a signaling cascade in which Syk functionsdownstream of Src and upstream of Cbl and other effectors associatedwith Cbl such as the p85 subunit of P1-3 kinase. The data demonstratedthat Src family kinases mediated the phosphorylation of Cbl in a Sykkinase independent manner in vivo. The data also revealed that Srcfamily kinases and Syk were required for phagocytosis mediated by thedownstream activation of PI-3 kinase.

Of note, more recent data identified the tyrosine residue at position731 of Cbl as a consensus binding site (YxxM; SEQ ID NO: 21) for the p85regulatory subunit of PI-3 kinase. Upon phosphorylation, this motif wasrecognized as a target for PTEN (data not shown). Hence, PTEN plays asignaling role in regulation of ITAMs action on PI-3 kinase to controlITAM signaling events.

Overexpression of PTEN in COS7 System Inhibits FcγIIA ITAM Signaling

Since tyrosine kinases are required for phagocytosis and lead to theactivation of PI-3 kinase which phosphorylates phospholipids that act assecond messengers, dephosphorylation of such phosphoinositides may servea role to downregulate this response. To address this issue, J774A.1 andCOS7 cells were genetically engineered to overexpress the dualspecificity phosphatase PTEN, which is known to dephosphorylate PIPS, acritical phosphoinositide second messenger. Overexpression of PTEN inCOS7 cells markedly inhibited phagocytosis of IgG coated sRBCs (FIG.12). PTEN expression reduced the phagocytic index (FIG. 12) by 95% ascompared to that of control cells. In contrast, the C124S mutant ofPTEN, which is catalytically dead and can act as a substrate trap,augmented the phagocytic index by a factor of 2.5. See FIG. 12. Thegreen bars represent the percent of the total cell population which wasphagocytic for at least one sRBC. These results demonstrated that PTENnegatively regulated IgG-mediated phagocytosis and that the catalyticactivity of PTEN was required for this suppression.

These data provide further evidence for the role of PTEN in theregulation of ITAM-mediated signaling in a variety of signaltransduction cascades essential for the propagation of the inflammatoryresponse in T cells, B cells and myeloid cells. Accordingly, suchbiological processes provide the basis for methods for screening andidentifying therapeutic agents which regulate these inflammatoryresponses.

Our observations suggest that the protein tyrosine kinases, Hck and Sykand PI-3 kinase are activated during FcγR mediated phagocytosis.Stimulation of macrophage cells with IgG coated particles activatesFcγRs, FcγRI and FcγRIII in turn activate Hck/Src kinase whichphosphorylates tyrosine in the ITAM motif in gamma chain associated withthese receptors. This provides a site for attachment and activation forSyk, which would activate the pathway downstream leading to subsequentactivatation of PI3-kinase which is needed for proper cytoskeletalassembly. On the other hand aggregation of FcγRIIB stimulatesphosphorylation of tyrosine in its ITIM motif to provide a site ofattachment and activation for phosphatases like PTEN and SHIP which inturn regulate the pathway downstream. Consistent with our data usingwortmannin, the 5′ inositide phosphatase, SHIP, serves as a negativeregulator of AKT phosphorylation and controls FcγR phagocytosis. Maedaet.al have provided evidence for the existence of two opposite signalingpathways upon aggregation of paired immunoglobulin receptors PIR-A andPIR-B (Maeda et al. (1998) J. of Exp. Med. 188:991) PIR-A induces thestimulatory signal by using ITAM in the associated γ chain while PIR-Bmediates the inhibitory signal through its ITIM. Our results areconsistent with the model that during Fcγ receptor mediated phagocytosisin J774A.1 mouse macrophages the FcγRI and FcγRIII utilize the ITAMmotif to send a positive signal inside the cell for phagocytic pathway;whereas FcγRIIB controls the pathway by starting a negative regulatoryloop via the ITIM motif through PTEN. Accordingly, Syk and Srcinhibitors should suppress ITAM immunoreceptor signaling and controlinflammation in concert with PI-3 kinase antagonists. It is also likelythat synergy will exist when agents which inhibit more than one ITAMpathway are combined in humans.

In summary, the data support the involvement of Syk, Src family kinasesand phosphatidyl inositol 3-kinase in positive regulation of Fcγreceptors mediated phagocytosis in our system. Described herein is thefirst evidence for the involvement of the protein/lipid phosphatase,PTEN in the negative regulation of phagocytosis and ITAM signaling.These data provide the basis for screening and identification oftherapeutic agents that modulate PTEN, PI-3 kinase or the signalingpathways downstream of PI-3 kinase (PDK-1, PAK, AKT, forkhead, etc).Inhibitors of ITAM propagated signals (e.g., 1) T cell receptor/CD3complex signaling in T cells; 2) B cell receptor signaling in B cells;3) ITAM receptor signaling including Fcγ receptors which mediate myeloidinflammatory diseases through the production of inflammatory cytokines,TNFa, IL-1, IL-4 etc.; 4) the FCεRI receptor in mast cells responsiblefor atopic disease and allergy; and 5) the FcαRI involved in mucosalallergic responses) should effectively regulate the immune responsepathway. Hence PTEN agonists and PI-3 kinase inhibitors shouldeffectively modulate immunopathologic states in animals. Likewise Sykand Src kinase inhibitors should effect ITAM myeloid signaling.

Example IV

PTEN and PI-3 Kinase Signaling Cascade Regulates p53 and Tumor-InducedAngiogenesis

As described in Examples I, II and III, PTEN regulates the tumor-inducedangiogenic response and thrombospondin expression in a malignant gliomamodel. A recent report by Sabbatini et al suggests a connection betweenthe PI-3 kinase cascade and the regulation of p53 signaling (Sabbatini,1999; J. Biol. Chem. 274:24263). Activation of PI-3 kinase/AKT pathwaysresults in the suppression of p53 dependent apoptotic pathways givingrise to conditions that are permissive for cell division. These datasuggest a molecular mechanism for the coordination of signals comingfrom growth factor receptors through PI-3 kinase cascades which wouldjointly regulate apoptosis, proliferation and recruitment of a new bloodsupply (neovascularization/angiogenesis). This signaling pathway appearsto be tightly regulated in normal tissues. During malignanttransformation, this coordinated regulation of cellular signaling islost. In the present example, we demonstrate that PTEN plays a role incoordinating these signaling events within the cell. We also show thatloss of PTEN leads to deregulation and tumor progression. Bearing inmind the link between PTEN phosphatase activity and P1-3 kinasesignaling pathways, we performed assays to determine whether P1-3 kinaseinhibitors were capable of reestablishing this regulatory feedbacksystem thereby restoring normal coordination of cell growth andangiogenesis.

Thrombospondin 1 (TSP-1), angiostatin, endostatin, tissue inhibitors ofmetalloproteinases (TIMPs) are potent inhibitors of angiogenesis(Dameron, et al., (1994) Science 265:1582; Good et al., (1990)PNAS87:6624). Malignant brain tumors are known to undergo a more robustangiogenic response as compared to their benign low-grade counterparts,and are classified histopathologically by the presence or absence ofhigh microvessel counts (microvessel density) (MVD). Regulation of PI-3kinase-dependent signals, including activation of AKT by VascularEndothelial cell Growth Factor and its receptors, the protein tyrosinekinases Flt-1 and KDR, have been implicated in brain tumor angiogenesis(Plate et al., (1992) Nature 359:845). Jiang et al demonstrated in thechicken chorioallantoic membrane model that PI-3 kinase-dependentpathways may regulate angiogenesis and VEGF expression in endothelialcells (Jiang et al., (2000) PNAS 97:1749). Immunohistochemical studiesin prostate tumor specimens have demonstrated that tumors containingPTEN mutations have higher microvessel counts than tumors expressingwild type PTEN (Giri et al., (1999) Hum. Pathol. 30:419).

To test the hypothesis that PTEN is connected to p53 transcription weperformed experiments in U87MG glioma cell lines which are wild type forp53 and deficient in PTEN. We conditionally expressed in these U87MGcells, wild type PTEN or catalytically defective mutants of PTEN todetermine if PTEN regulates p53 transcription. We then performedexperiments with the kinase inhibitor using the parental U87MG cells todetermine if LY294002 control over PIP₃ metabolism would prevent tumorgrowth and block angiogenesis in vivo.

The following protocols are provided to facilitate the practice ofExample IV.

The constructs and encoding the PTEN mutants have been previouslydescribed in Example I. Tumor implantation methods are also provided inthe previous examples.

Treatment of Mice with LY294002.

LY294002 was administered at a dosage of 100 mg/kg delivered daily byintraperitoneal injection in a small volume of 100% DMSO for 2 weeks,beginning 2 days after tumor implantation. No untoward effects werenoted in mice treated with either LY294002 or DMSO. Control mice wereinjected with small volume (10 ml) of 100% DMSO. Daily measurement oftumor volume was performed in 3 coordinates using calipers.

Biochemical Analysis.

Immunoblots were performed on cell lysates obtained from U87 cells grownin tissue culture or from multiple cryostat sections of subcutaneoustumor tissues. A Bradford assay was performed to determine proteinconcentration of each lysate. Equivalent amounts of protein wereresolved by SDS PAGE and transferred to nitrocellulose. Membranes wereprobed with antisera specific for PTEN, AKT, phospho-S473-AKT. We used awell characterized mdm2 promoter linked to firefly luciferase (mdm2lucinserted into the pGL2 vector) that contains p53 DNA binding elements(Ouchi et al., (1998) PNAS 95:2302) to study p53 specific transcriptionin U87 cells under muristirone induced PTEN expression conditions.Another construct, pGL2, contained the mdm2luc promoter which is deletedfor p53 response element was used as a negative control. Cells werecotransfected with pRSVβgal to normalize mdm2 luciferase activity fortransfection efficiency. The Tropix-galacto-light kit and Promegaluciferase assay system was used to quantitate β-galactosidase andluciferase activity, respectively.

Immunohistochemical and Histopathology.

Microvessel density (MVD) was determined for as described in theprevious examples.

Results

The previous examples demonstrated that the muristirone inducibleexpression of PTEN in U87 cells results in increased levels ofthrombospondin 1 expression, a negative regulator of angiogenesis. Inthe present example, we demonstrate that wild type PTEN suppressed theactivation of phospho-AKT without affecting total AKT (FIG. 13). Theinduced expression of mutant G129R had no effect on phosphoAKT (FIG.13). The data presented in the previous examples indicate that PTEN mayregulate angiogenesis through the induction of TSP-1. One transcriptionfactor that upregulates TSP-1 is the tumor suppressor protein p53(Dameron, (1994) Science 265:1582). We sought to determine if there wasa link between PTEN, TSP1 and p53.

The data presented herein demonstrate that PTEN regulates p53transcription (FIG. 14). The induction of wild type PTEN and not mutantPTEN induced p53 dependent transcription in U87 cells (7.5-foldinduction) (FIG. 14). Muristirone induced expression of wild type PTENor G129R protein was equivalent whereas G129E induction was slightlygreater (see insert, FIG. 14). Controls were performed using anmdm2luciferase construct deleted in critical p53 binding sites toconfirm the specificity for PTEN induction of p53 specifictranscription. These data provide compelling evidence that PTEN and p53are linked in a common pathway and therefore provide new biochemicaltargets for influencing PTEN regulation of expression of TSP-1 throughthe regulation of p53 transcription.

To determine whether PI-3 kinase exerts control over angiogenesis or thegrowth of glial tumors in vivo the orthotopic brain tumor modeldescribed in Example I was utilized. To assess the effect of LY294002 onangiogenesis, we treated mice with LY294002 (100 mg/kg/dose×2 weeks) orDMSO as negative control. Tumor volumes were recorded daily (FIG. 15).On day 14 we stained cryostat sections from subcutaneous tumors for CD31(PECAN). CD31 is an endothelial marker used to measure the microvesseldensity of these tumors. Microvessel density was assessed from multipledigitized images of CD31-stained tumor tissue at 100× magnification (3fields were evaluated per tumor) and counted blindly for the number ofCD31 positive microvessels per unit surface area as described (Weidneret al., (1991) N. Engl. J. Med. 324:1). Quantitation of microvesseldensity in tumors treated with DMSO versus LY294002 are shown in FIG.16. Compared to controls (FIG. 16), it was observed that LY294002markedly suppressed the tumor-induced angiogenic response in this model(MVD is 22±5 in LY294002 treated tumors versus 50±6 in the controls).Importantly, microvessel determinations were performed on day 7 afterimplantation to compare angiogenic activity of tumors of similar size.At the time of analysis the tumors were approximately 400 mm3 in thecontrol and approximately 35 mm3 in the LY294002 treated mice. Thesedata argue against an effect of tumor hypoxia or size on the inductionof angiogenesis. It is likely that the effects of LY294002 are complexand that the size of tumor mass may contribute at later time points tothe induction of angiogenesis. Despite this caveat, the data demonstratethat LY294002 dramatically suppressed the angiogenic response of U87MGcells in vivo.

We also examined the effect of LY294002 on tumor growth and incidence ofbrain tumor in a nude mouse model. See FIG. 17. In a previous study, invivo activity of LY294002 against an ovarian carcinoma was observed.However, it is difficult to interpret these results as the tumor wasgrown as an ascitic tumor and LY294002 was injected into peritonealcavity. In other words, the carcinoma was not assessed in its naturalmilieu. Hu et al., Clin. Can. Res. 6:880). Our results show conclusivelythat subcutaneous tumor growth is markedly suppressed by LY294002treatment (FIGS. 15 and 17). In additional experiments, we observed thatLY294002 markedly suppressed the intracranial growth of U87MG cells innude mouse model. In control mice treated with DMSO, 4/5 had grosslyvisible and/or histologically confirmed brain tumors by H & E analysisby day 25 after implantation, whereas in 0/5 mice treated with LY294002had grossly detectable or histologic evidence of intracranial tumor whenexamined on day 42. Other recent reports suggest that LY294002 and PTENreconstitution may increase tumor responsiveness to DNA damage fromchemotherapy and radiation. Our results suggest that PTEN exerts itsregulatory effects through the induction of p53 transcription, a knownfactor in the induction of apoptosis.

Recent in vitro data suggest a link between PI-3 kinase and downstreamtargets including AKT, HIF1α and VEGF in the potential control ofangiogenesis. In the present example, we show that the PTEN tumorsuppressor controls p53 transcription in U87 glioblastoma cells. Hollandet al recently reported that the introduction of activated AKT and Rasinto glial cells of the mouse brain results in the development ofglioblastomas (Holland et al., (2000) Nat. Genet. 25:55). Thus, thecombined data indicate that inhibitors of PI-3 kinase and downstreamtargets such as AKT should provide therapeutic efficacy in the treatmentof malignant gliomas. Finally, the results presented herein provide thefirst evidence that LY294002 controls tumor-induced angiogenesis througha mechanism that appears to involve the regulation of p53 transcription.Accordingly, these data support the hypothesis that these two tumorsuppressor genes are localized on the same signaling pathway for thecoordinated control of angiogenesis in tumor cells.

Example V PTEN Reconstitution Enhances Sensitivity of Tumors toChemotherapy and Radiation Therapy in Vitro and in Vivo

The data presented in Example IV demonstrate that a molecular linkexists between the activities of PTEN and p53 tumor suppressor genes,p53-mediated transcription and phosphorylation of MDM2 by AKT. Thesedata indicate that activation of the PI-3 kinase pathway can becorrelated with a reduction of programmed cell death leading to a higherrisk of tumor progression. Thus, agents which modulate the PTEN pathwaycan be used in certain cell populations to influence the apoptoticmechanisms triggered by chemotherapy and radiation and other cellularstresses. PI-3 kinase, MDM2 and AKT as well as PTEN provide idealbiological targets for the development of such agents. Thus, the presentinvention provides methods for identifying and biochemicallycharacterizing small molecules which modulate the biological processesregulated by these proteins, including, but not limited to apoptosis,proliferation, differentiation and chemosensitivity.

The data set forth in this example implicate a role for PTEN in chemo-and radiosensitivity which influences tumor-induced angiogenesis and p53tumor suppressor activity. Since PTEN is mutated or deleted in 50% ofall glioblastoma, we assessed whether such cells are rendered sensitiveto chemotherapy upon stable introduction of wild-type PTEN. U87MGglioblastoma cells, null for PTEN, or glioblastoma cells stablyexpressing catalytically inactive PTEN (R130M cells), required 4.7 μM ofetoposide to achieve a 50% cell kill. Glioblastoma cells engineered tostably express wild-type PTEN were sensitized requiring 0.047 μM ofetoposide to achieve a 50% cell kill, a 100-fold increase insensitivity. See FIGS. 18 and 19. Compensation for the absence of PTENin the parental line of glioblastoma was achieved by treatment of thecells with LY294002. This agent rendered the parental, PTEN-null cellsas sensitive to etoposide as cells overexpressing PTEN. Theseobservations show that PTEN and inhibitors of PI 3-kinase sensitizecancers to chemotherapy by blockade of PI 3-kinase/Akt survivalsignaling, thereby permitting the p53 tumor suppressor to transmit itsdeath signal.

The following protocols are provided to facilitate the practice ofExample VI.

Materials.

The D0p1 antibody to p53 was from Santa Cruz Biotechnology.

Cell Culture, Treatments, and Transfections.

U87MG cells, breast adenocarcinomas, MCF-7 cells, T47D breast ductalcarcinoma cells, 293T human embryonic kidney cells transformed withadenovirus, and H1299 non-small lung .carcinoma cells were cultured inDMEM supplemented with 10% fetal bovine serum at 37° C. under 5% CO₂.Stable clones of U87MG and U373MG glioma cells were generated usingretroviral vectors pBABEpuro to express wild type and mutants of PTEN.

The MDM2 promoter with and without p53 response elements was operablylinked to a nucleic acid encoding luciferase as described in Example IV.Luciferase assays were conducted twenty-four hours after transienttransfection using the luciferase assay system and the galacto-light kitto assay β-gal activity.

MTT assays. Cells were seeded (6000/well) into a 96 well plate.Twenty-four hours later the growth medium was changed to 1% serum andother reagents were added as described. Treatments proceeded for 24-72h. At each time point the MTT assay was performed. MTT (10 ul of a 5mg/ml stock solution dissolved in RPMI medium) is added to each well ofcultures 4 hours before quantitation of viable cell number by measuringabsorbance in each well at 650 nm with a microplate reader (MolecularDevises,)

Agents added to cell LY294002 alone or in combination with etoposide. Inaddition, comparison was made between U87MG cells null for PTEN andreconstituted with PTEN protein. The absorbance of each well wasmeasured at 570 nM with a microplate reader. The amount of absorbance isa reflection of mitochondrial activity of cells and hence is a methodfor the quantitation of viable cell numbers in multiple replicates.

Effect of PTEN Expression on Radiosensitivity.

To determine the role of PI-3 kinase pathways and PTEN in control ofradiation sensitivity, U87MG reconstituted with wild type or mutant(G129R) forms of PTEN under constitutive or muristirone inducedconditions were exposed in vitro to a gamma irradiation source of 12 Gy,20 Gy, 30 Gy, 40 Gy, 50 Gy at 1.2 Gy per minute as a single dose.

Cells were then washed gently and plated in tissue culture 96 well plateand at different time intervals after radiation exposure viable cellnumbers were determined using MTT assay. An IC50 was established forcells expressing PTEN or treated with 5-10 uM LY294002. The results aresimilar to those observed with etoposide sensitivity in that PTENreconstitution or LY294002 dramatically increased the radiationsensitivity of this p53 wild type tumor in vitro. Evaluation in vivoshowed similar results as described for our experiments forantiangiogenic and antimetastatic effect of LY294002 or PTENreconstitution.

The data reveal that the induction of p53 associated with PTEN wasoperative in the induction of a radiosensitive state.

The clones utilized in this example express physiologic levels of PTENthereby more closely approximating biological reconstitution of PTENrather than overexpression of the protein. In the studies described inthe prior art and the previous examples, PTEN is overexpressed andgenerates cells which are growth arrested by virtue of excess levels ofPTEN. PTEN expression was assessed in the present U373MG clones byWestern blot and found to be equal to or less than levels of PTENprotein measured in whole brain lysates or cultivated human primaryastrocytes. Ideally, assays for assessing PTEN function should be basedon experimental conditions wherein PTEN expression levels accuratelyreflect the levels observed in normal cells. It is known that PTENlevels are closely regulated by endogenous biochemical feedbackmechanisms under normal conditions thereby allowing cells to undergoregulated proliferation in response to proper physiological stimuli.Thus, PTEN functions in normal cells as a rheostat to control vital PIP₃linked functions.

As demonstrated herein, PTEN activity influences the sensitivity ofU87MG cells to genotoxic stress induced by the topoisomerase inhibitor,etoposide (VP16) and radiation. Exposure of U87MG cells to etoposide inthe presence of PI-3 kinase inhibitor, LY294002 dramatically shifted thedose/response curve towards a more sensitive state. Similarly PTENreconstituted tumor cells were markedly more sensitive to etoposideinduced cell death and radiation. Thus, PTEN activity modulates p53function thereby regulating and coordinating p53 levels in the cell.Accordingly, activation of PTEN and the PI-3 kinase pathway sensitizescells to p53 mediated cell death through the control of p53 inducedapoptosis.

The data reveal that LY294002 dramatically sensitizes the parental U87MGcells to the cytotoxic effect of etoposide (VP-16) as shown by MTT assayat a concentration where LY294002 has no effect on cell viability. Theconcentration of etoposide utilized demonstrates minimal cytotoxicactivity (1 uM) while the combination of etoposide and LY294002 appearsto act synergistically giving rise to enhanced cytotoxicity. There is a5-fold increase in cytotoxicity observed in cells pretreated with thePI-3 kinase inhibitor. These results have been repeated 20 times and arehighly statistically significant (p<0.0001) and show that LY294002induces a chemosensitive state in this p53 wild type glioma cell line.Similar data were obtained for effect of PTEN reconstitution in thissystem and in other tumor cell lines. PTEN reconstitution or LY294002induced a marked sensitivity of tumor cells to etoposide, adriamycin,cytoxan, asparaginase, vincristine, busulfan and other chemotherapeuticagents. In addition LY294002 and PTEN reconstitution induce a markedsensitivity to ionizing radiation using the same methodology.

LY294002 and PTEN reconstitution also induce a sensitivity to apoptosisinduced by other environmental stress including: osmotic, endotoxicstress, nutritional stress, metabolic stress, hyperoxic, hypoxic,chemical, temperature, immunologic, other forms of electromagneticradiation, heat shock, using the same methods for enablement. Theinhibition of PTEN reduced the apoptotic response to each of these formsof cellular stress in vivo and in vitro.

While certain preferred embodiments of the present invention have beendescribed and specifically exemplified above, it is not intended thatthe invention be limited to such embodiments. Various modifications maybe made to the invention without departing from the scope and spiritthereof as set forth in the following claims.

1. A method for identifying agents which modulate PTEN activity,comprising: a) providing a host cell wherein PTEN is expressed; b)contacting said host cell with a test agent suspected of modulating PTENactivity; c) assessing said modulation as a function of alterations inactivated AKT levels in the presence of said agent.
 2. A method asclaimed in claim 1, wherein said PTEN is mutated.
 3. A method as claimedin claim 2, wherein said mutation in PTEN is selected from the groupconsisting of G129E, G129R, R130M and C124S.
 4. A method as claimed inclaim 2, wherein said PTEN is inactive and said test agent suppressesconcomittant AKT activating activity.
 5. A test agent identified by themethod of claim
 4. 6-88. (canceled)